CA3215623A1

CA3215623A1 - Flavonoid and anthocyanin bioproduction using microorganism hosts

Info

Publication number: CA3215623A1
Application number: CA3215623A
Authority: CA
Inventors: Jingyi Li; Nicholas BRIDEAU; Joshua BRITTON; Erik Holtzapple
Original assignee: Debut Biotechnology Inc
Current assignee: Debut Biotechnology Inc
Priority date: 2021-04-13
Filing date: 2022-04-13
Publication date: 2022-10-20
Also published as: AU2022256988A9; KR20230170713A; US20220333123A1; BR112023021282A2; US20220333087A1; IL307636A; CN117413066A; WO2022221392A1; AU2022256988A1; US20220333118A1; JP2024513971A; EP4323528A1; US20220333117A1; US20220333122A1

Abstract

The invention is directed to methods involved in the production of flavonoids, anthocyanins and other organic compounds. The invention provides cells engineered for the production of flavonoids, anthocyanins and other organic compounds, where the engineered cells include one or more genetic modifications that increase flavonoid production by increasing metabolic flux to flavonoid precursors and/or reducing carbon losses resulting from the production of byproducts.

Description

2 FLAVONOID AND ANTHOCYANIN BIOPRODUCTION USING MICROORGANISM
HOSTS
I. RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application No.
63/174,403, filed on April 13, 2021. The content of U.S. Provisional Application No. 63/174,403 is hereby incorporated by reference in its entirety.
SEQUENCE LISTING
This application contains a sequence listing filed in electronic form as an ASCII.txt file entitled DEBU-009-01-WO-Sequence-Listing.txt, created on March 21, 2022, last modified April 12, 2022, and having a size of 448 KB. The content of the sequence listing is incorporated herein its entirety.
III. FIELD OF THE INVENTION
The invention related to materials (including engineered cells and cell lines) and methods involved in the production of flavonoids, anthocyanins and other organic compounds.
IV. BACKGROUND OF THE INVENTION
Flavonoids and anthocyanins are natural products produced in plants that find a variety of roles such as antioxidants, ultraviolet (UV) defense mechanisms, and colors.
Over the past several years, the health benefits of flavonoids and anthocyanins have been widely demonstrated.
These compounds are capable of scavenging radicals and can act as enzyme inhibitors and anti-inflammatory agents. With these recognized health and color benefits, much research has gone into understanding how these compounds are made in nature. Flavonoids and anthocyanins are synthesized from phenylpropanoid starter units and malonyl-Cofactor-A (malonyl-CoA) extender units that then undergo modifications to create many polyphenol compounds such as taxifolin, naringenin, and (+)-catechin. However, in most cases, these compounds are extracted or chemically manufactured.

V. SUMMARY OF THE INVENTION
To move away from agriculture and chemically derived products, we have created engineered cells for the bioproduction of flavonoids and anthocyanins. This approach provides a feasible route for the rapid, safe, economical, and sustainable production of a wide variety of important flavonoids.
Herein, a range of flavonoids and anthocyanins including naringenin, eriodictyol, taxifolin, dihydrokaempferol, (+)-catechin, cyanidin, and cyaninidin-3-glucoside are biomanufactured using a modified microbial host. Herein, the engineered cells include one or more genetic modifications that increase(s) flavonoid and anthocyanin bioproduction by increasing metabolic flux to flavonoid precursors and/or reducing carbon losses resulting from the production of byproducts.
Provided herein are cells engineered for the production of flavonoids, anthocyanins and other organic compounds, where the engineered cells include one or more genetic modifications that increase flavonoid production by increasing metabolic flux to flavonoid precursors and/or reducing carbon losses resulting from the production of byproducts. As nonlimiting examples, a genetic modification can be a modification for over-expressing or under-expressing one or more endogenous genes in the engineered host cell or can be a modification for expressing one or more non-native genes in the engineered host cell. Engineered cells as provided herein can include multiple genetic modifications.
Also provided are cell cultures for producing one or more flavonoids or anthocyanins.
The cell cultures include engineered cells as disclosed herein in a culture medium that includes a carbon source that can also be an energy source, such as glycerol, sugar, or an organic acid. In various embodiments, the culture medium can include at least one feed molecule such as but not limited to one or more organic acids or amino acids that can be converted into a flavonoid precursor (such as tyrosine, p-coumaroyl-CoA or malonyl-CoA). Examples of feed molecules include, but are not limited to, acetate, malonate, tyrosine, phenylalanine, pantothenate, coumarate, etc. In some embodiments, the feed molecules may be of reduced or low purity. For example, glycerol as a feed molecule may be crude glycerol, including a biomass comprising glycerol, for example, glycerol obtained as a byproduct of biodiesel processing. Alternatively, or in addition, the culture medium can include a supplemental compound that can be a cofactor or a precursor of a cofactor used by an enzyme that functions in a flavonoid pathway, such as, for examples, bicarbonate, biotin, thiamine, pantothenate, alpha-ketoglutarate, ascorbate, or 5-aminolevulinic acid.
Further provided are methods for producing flavonoids and anthocyanins that include culturing a cell engineered for the production of flavonoids or anthocyanins as provided herein under conditions in which the cell produces flavonoids or anthocyanins. In some examples, the methods include culturing the engineered cells in a culture medium that includes at least one feed molecule or supplement such as but not limited to: tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid. The methods can further include recovering at least one of the flavonoids from culture medium, whole culture, or the cells.
In a first aspect, provided herein are cells engineered to produce one or more flavonoids or anthocyanins, wherein the cells include, in addition to nucleic acid sequences encoding either tyrosine ammonia lyase activity and/or phenylalanine ammonia lyase activity and cinnamate-4-hydroxylase activity, 4-coumarate-CoA ligase activity, chalcone synthase activity, chalcone isomerase activity, flavanone-3-hydroxylase activity, flavonoid 3'-hydroxylase activity or flavonoid 3'5'-hydroxylase activity, cytochrome P450 reductase activity, leucoanthocyanidin reductase activity, and dihydroflavono1-4- reductase activity, one or more genetic modifications for improving production of the flavonoids or anthocyanins. As set forth herein, a cell that is engineered to produce one or more of the flavonoids is engineered to include an exogenous nucleic acid sequence encoding tyrosine ammonia lyase activity that can form 4-coumaric acid using tyrosine as substrate (e.g., tyrosine ammonia lyase TAL, EC: 4.3.1.25) or, alternatively or in addition, an exogenous nucleic acid sequence encoding phenylalanine ammonia lyase activity that can convert phenylalanine to trans-cinnamic acid and an exogenous nucleic acid sequence encoding cinnamate-4-hydroxylase activity that forms 4-coumaric acid from trans-cinnamic acid, an exogenous nucleic acid sequence encoding CoA ligase activity that forms p-coumaroyl-CoA
from coumaric acid (e.g., 4-coumarate--CoA ligase, 4CL, EC:6.2.1.12), an exogenous nucleic acid sequence encoding polyketide synthase activity that forms naringenin chalcone using malonyl-CoA and p-coumaroyl-CoA as substrates (e.g., chalcone synthase, CHS, EC:2.3.1.74), an exogenous nucleic acid sequence encoding chalcone isomerase activity that forms naringenin

3 from naringenin chalcone via its cyclase activity (e.g., chalcone-flavonone isomerase, CHI, EC:5.5.1.6), an exogenous nucleic acid sequence encoding flavanone-3-hydroxylase activity that forms dihydrokaempferol from naringenin or forms taxifolin from eriodictyol (e.g., naringenin 3-dioxygenase, F3H, EC: 1.14.11.9), an exogenous nucleic acid sequence encoding flavonoid 3'-hydroxylase or flavonoid 3'5'-hydroxylase activity coupled with an exogenous nucleic acid sequence encoding cytochrome P450 reductase activity to form taxifolin or dihydromyricetin from dihydrokaempferol or to form eriodictyol or pentahydroxyflavone from naringenin (e.g., flavonoid 3'-monooxygenase, F3'H, EC: 1.14.13.21, EC: 1.14.14.82; cytochrome -P450 reductase, EC:1.14.14.1; F3'5'H, EC:1.14.14.81 ), an exogenous nucleic acid sequence encoding dihydroflavono1-4-reductase activity that forms leucocyanidin from taxifolin, leucodelphinidin from dihydromyricetin, orleucopelargonidin from dihydrokaempferol (e.g., dihydroflavonol 4-reductase, EC:1.1.1), and an exogenous nucleic acid sequence encoding leucoanthocyanidin reductase activity that forms catechin from leucocyanidin (e.g., leucoanthocyanidin reductase, LAR, EC:1.17.1.3). Optionally, a cell that is engineered to produce anthocyanins is further engineered to include an exogenous nucleic acid sequence encoding anthocyanin synthase activity that forms cyanidin from catechin or leucocyanidin, forms delphinidin from leucodelphinidin, or forms pelargonidin from leucopelargonidin (e.g., anthocyanin synthase, ANS, EC:1.14.20.4) and to include an exogenous nucleic acid sequence encoding glucosyltransferase activity that forms cyanidin-3-0-beta-D-glucoside from cyanidin, delphinidin-3-0-beta-D-glucoside from delphinidin, or pelagonidin-3-0-beta-D-glucoside from pelagonidin (e.g., anthocyanidin 3-0-glucosyltransferase, 3GT, EC :2.4.1.115).
The cells provided herein that are engineered to produce flavonoids or anthocyanins are further engineered to increase the production of flavonoids or anthocyanins product, for example by increasing metabolic flux to a flavonoid or anthocyanin pathway, or by decreasing byproduct formation.
A cell engineered to produce a flavonoid is further engineered to increase the supply of precursor malonyl-CoA. One strategy for increasing malonyl-CoA includes increasing acetyl-CoA carboxylase (ACC) activity. In various embodiments, the ACC enzyme, which in most eukaryotes, including fungi, is a large single chain polypeptide, and in plant and bacteria such as E. colt is a multi-subunit enzyme, is overexpressed in the host strain.
Examples of acetyl-CoA
carboxylase that can be expressed in a host cell engineered to produce a flavonoid or anthocyanin include, without limitation, the ACC genes of Mucor circinelloides, Rhodotorula

4 toruloides, Lipomyces starkeyi, Ustilago maydis, and orthologs of these ACCs in other species having at least 50% amino acid identity to these ACCs.
Additional strategies for increasing malonyl-CoA include increasing acetyl-CoA, which is converted to malonyl-CoA by acetyl-CoA carboxylase (ACC). In some embodiments, acetyl-CoA synthase (ACS) that converts acetate and CoA to acetyl-CoA is over-expressed in the host cells. Cultures of engineered host cells that include overexpressed nucleic acid sequence encoding ACS can optionally include acetate in the culture medium. Examples of acetyl-CoA
synthase that can be expressed in a host cell engineered to produce a flavonoid or anthocyanin include, without limitation, the ACS gene of E. coil, the ACS of Salmonella typhimurium, orthologs of these ACSs in other species having at least 50% amino acid identity to these ACSs.
Also considered, in further embodiments, is an engineered host cell that overexpresses a gene encoding pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA. Further, in E. coli, a variant of the Lpd subunit of PDH can be expressed that includes a mutation (E354K) that reduces inhibition of PDH by NADH.
Alternatively, or in addition to strategies for increasing ACC activity and strategies for increasing acetyl-CoA, strategies for increasing malonyl-CoA by mechanisms that do not rely on the activity of an ACC can be employed. In some embodiments, a cell engineered to produce a flavonoid, or an anthocyanin, is further engineered to increase the cell's supply of malonyl-CoA
includes an exogenous nucleic acid sequence encoding a malonyl-CoA synthetase that generates malonyl-CoA from malonate. Examples of malonyl-CoA synthetases include the malonyl-CoA
synthetases of Streptomyces cod/color, Rhodopseudomonas palustris, or a malonyl-CoA
synthetase having at least 50% identity to any of these or other naturally occurring malonyl-CoA
synthetases. Malonate can optionally be added to the culture medium of a culture that includes a cell engineered to express a malonyl-CoA synthetase. An engineered cell that includes an exogenous gene encoding a malonyl-CoA synthetase can also include an exogenous nucleic acid sequence encoding a malonate transporter, such as a malonate transporter encoded by a matC
gene, for example, of Streptomyces cod/color, or a malonate transporter encoded by DctPQM of Sinorhizobium medicae .
In additional embodiments, a cell engineered to produce a flavonoid or an anthocyanin is further engineered to include an exogenous nucleic acid sequence encoding malonate CoA-

5 transferase that makes malonyl-CoA by direct transfer of the CoA from acetyl-CoA. Examples of malonate CoA-transferase that can be expressed in an engineered cell as provided herein include, without limitation, the alpha subunit (mdcA) of malonate decarboxylase from Acinetobacter calcoaceticus, Geobacillus sp, or a transferase having at least 50% identity to any of these or other naturally occurring malonate CoA-transferases.
In some embodiments, a cell engineered to produce flavonoids or anthocyanins is further engineered to increase the supply of coenzyme A (CoA) to increase its availability for producing acetyl-CoA, malonyl-CoA, and/or p-coumaroyl-CoA. Strategies for increasing CoA
supply include upregulating endogenous pantothenate kinase (PanK) (EC:2.7.1.33) that produces CoA
from pantothenate. Alternatively, or in addition, a host cell can be engineered to include a nucleic acid sequence encoding type III pantothenate kinase that is not feedback inhibited by coenzyme A, such as CoaX gene ofpseudomonas aeruginosa (EC:2.7.1.33). Cultures of cells engineered for the production of flavonoids or anthocyanins can in some embodiments include a medium that includes pantothenate, a precursor of CoA biosynthesis, and can optionally also include cysteine, used in the CoA biosynthesis.
Additional strategies to increase malonyl-CoA flux to the flavonoid pathway include mutation or downregulation of one or more genes that function in fatty acid biosynthesis.
Without limiting the embodiments to any particular mechanism, limiting fatty acid biosynthesis can increase the malonyl-CoA supply available for flavonoid biosynthesis. In some embodiments, the gene beta-ketoacyl-ACP synthase II (E. coil fabF) can be disrupted to reduce fatty acid biosynthesis. Another example of a fatty acid biosynthesis gene of a host cell that may be mutated or downregulated is a gene encoding malonyl-CoA-ACP transacylase (E. coil fabD).
Other fatty acid biosynthesis genes of the engineered host cell that can be downregulated include a beta-ketoacyl-ACP synthase I enzyme E. coil fabB) and acyl carrier protein (E. coil acpP).
Additional genetic modifications that may be present in a host cell engineered to produce flavonoids or anthocyanins include downregulation, disruption, or deletion of genes encoding alcohol dehydrogenase, lactate dehydrogenase, pyruvate oxidase, acetyl phosphate transferase and acetate kinase. In an E. coil host cell, genes that are downregulated, disrupted, or deleted can include aldehyde-alcohol dehydrogenase (adhE), lactate dehydrogenase (ldhA), pyruvate oxidase (poxB), and enzyme acetate kinase phosphate acetyltransferase (ackA-pta).

6 Further, a cell engineered for the production of flavonoids or anthocyanins can have one or more genes encoding thioesterases downregulated, disrupted, or deleted to prevent hydrolysis of precursors malonyl-CoA, actetyl-CoA, and/or p-coumaryol-CoA. For example, in an E. coli host one or more of the thioesterase genes tesA, tesB, yciA, and ybgC can be downregulated, disrupted, or deleted.
Alternatively, or in addition, genes encoding enzymes of the tricarboxylic acid cycle (TCA), such as succinate dehydrogenase, can be disrupted or downregulated to increase alpha-ketoglutarate supply which serves as a cofactor for one or more of the flavonoid and anthocyanin pathway enzymes. Other TCA enzymes that can be downregulated include citrate synthase that converts acetyl-CoA to citrate.
Also considered, in further embodiments, is an engineered host cell for the production of flavonoids or anthocyanins to upregulate the endogenous biosynthesis of amino acid tyrosine.
Tyrosine is one of the precursors for the flavonoid biosynthesis and its conversion to coumaric acid is the first committed step of the pathway. L-tyrosine is one of the three aromatic amino acids derived from the shikimate pathway. The initial step of the shikimate pathway is catalyzed by DAHP synthase isozymes and regulated through feedback-inhibition.
Strategies to increase tyrosine production can include, without limitation, transcriptional deregulation, removing feedback inhibition, overexpression of rate-limiting enzymes, and/or deletion of the L-phenylalanine branch of the aromatic acid biosynthetic pathway. For example, in an E. coil host the tyrR gene can be disrupted, feedback-inhibition-resistant versions of the DAHP synthase (aroG) and chorismate mutase (tyrA) can be introduced, and/or rate-limiting enzymes, shikimate kinase (aroK or aroL) and quinate (QUIN)/shikimate dehydrogenase (ydiB) can be overexpressed. Further, the Phosphoenolpyruvate synthase (ppsA) and transketolase (tktA) can be exogenously introduced to enhance tyrosine production.
Also considered, in further embodiments, is an engineered host cell for the production of flavonoids or anthocyanins further engineered to upregulate the endogenous biosynthesis of cofactor heme. Cytochrome P450 (CYPs), one of the exogenous genes in the engineered cells provided herein, contain heme as a cofactor. Improving heme supply can be an effective strategy to increase flavonoid biosynthesis. 5-aminolevulinic acid (ALA) is the first committed precursor to the heme pathway. Strategies to increase heme supply include overexpression of the genes that

7 synthesize the precursor ALA. In an E. coil host, ALA is formed from the 5-carbon skeleton of glutamate (the C5 pathway). The three enzymes involved in ALA biosynthesis are glutamyl-tRNA synthetase (g1tX), glutamyl-tRNA reductase (hemA), and glutamate-l-semialdehyde aminotransferase (hemL). In an E. coil host, the engineered cells provided herein can be further engineered to express or overexpress hemA or its variants, and/or hemL to increase the heme precursor ALA production. The nonlimiting examples of hemA gene that can be overexpressed include a mutated hemA (inserting two lysine residuals between Thr-2 and Leu-3 at N terminus of hemA gene from Salmonella typhimurium (EC:1.1.1.70). Alternatively, or in addition, a heterologous ALAS gene can be introduced to produce ALA via the C4 pathway (ALS is synthesized by the condensation of glycine and succinyl-CoA). Nonlimiting examples of heterologous ALAS that can be expressed in E. coil include ALAS of Bradyrhizobium japonicum (EC: 2.3.1.37), ALAS of Rhodobacter capsulatus, or an ALAS having at least 50%
sequence identity to a naturally occurring ALAS. Further, one or more of the downstream genes (e.g., in E. coil hemB, hemC, hemD, hemE, hemF, hemG, hemI, or hemH) that catalyze the synthesis of heme from ALA can be overexpressed to drive the flux from ALA to heme production. Cultures of cells engineered for the production of flavonoids or anthocyanins can in some embodiments include a medium that includes succinate and/or glycine, precursors of heme biosynthesis via the C4 pathway.
In another aspect, provided herein are cell cultures that include engineered cells as provided herein in a culture medium, where the culture medium includes a carbon source that is also an energy source for the cells, where the carbon source can be, for example, glycerol, a sugar, or an organic acid, as nonlimiting examples. The culture medium can further include a feed molecule that is used to produce flavonoids or anthocyanins. A feed molecule can be, for example, acetate, malonate, tyrosine, pantothenate, coumarate, biotin, alpha-ketoglutarate, ascorbate, 5-aminolevulinic acid, succinate, or glycine. In some embodiments, the culture comprises a culture medium that includes a carbon source and at least one supplement that is a cofactor of an enzyme or is a precursor of an enzyme cofactor.
In yet another aspect, methods for producing flavonoids and anthocyanins that include incubating a culture of engineered host cell as provided herein to produce flavonoids or

8 anthocyanins. The methods can further include recovering at least one of the flavonoids from the cells, the culture medium, or the whole culture.
In yet another aspect, the invention provides an engineered host cell that comprises one or more genetic modifications resulting in production of flavonoid or anthocyanin from a carbon source that can also be an energy source, through multiple chemical intermediates, by the engineered host cell. In certain embodiments, the production of flavonoid or anthocyanin from glycerol occurs through enzymatic transformation. In certain embodiments, the production of flavonoid or anthocyanin from a carbon source that can also be an energy source occurs through enzymatic transformation. In certain embodiments, the carbon source is selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol; and (vi) any combination thereof In certain embodiments, the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol;
and (vi) any combination thereof In certain embodiments, one or more genetic modifications lead to increase metabolic flux to flavonoid precursors or cofactors. In certain embodiments, one or more genetic modifications cause reduction of formation of byproducts. In certain embodiments, one or more genetic modifications are selected from: (i) one or more modifications for over-expressing one or more endogenous genes in the engineered host cells;
(ii) one or more modifications for under-expressing one or more endogenous genes in the engineered host cells; (iii) one or more genetic modification is expressing one or more non-native genes in the engineered host cells; and (iv) a combination thereof. In certain embodiments, the engineered host cell is cultured in a medium comprising molecules selected from: tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid, wherein one or more of the selected molecules are the chemical intermediates, including molecules in the biosynthesis pathway or cofactors. In certain embodiments, the engineered host cell comprises at least one or more nucleic acid sequences selected from: (i) nucleic acid sequences encoding tyrosine ammonia lyase activity; (ii) nucleic acid sequences encoding phenylalanine ammonia lyase activity; (iii) nucleic acid sequences encoding cinnamate 4-hydroxylase activity; (iv) nucleic acid sequences encoding 4-courmarate-CoA ligase (4CL) activity; and (v) any combination thereof In certain embodiments, the engineered host cell comprises at least one or more peptides selected from: (i)

9 chalcone isomerase; (ii) chalcone synthase; (iii) a fusion protein comprises a chalcone synthase and a chalcone isomerase; and (iv) any combination thereof. In certain embodiments, the engineered cell is E. coil. In certain embodiments, one or more genetic modifications decreases fatty acid biosynthesis. In certain embodiments, the engineered host cell comprises an exogenous nucleic acid sequence selected from: (i) nucleic acid sequence encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (v) any combinations thereof In certain embodiments, the engineered host cell comprises an exogenous nucleic acid sequence selected from the group consisting of: (i) nucleic acid sequences encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acid sequence encoding 4-courmarate-CoA ligase activity, wherein 4-courmarate-CoA ligase forms p-coumaroyl-CoA from coumaric acid (v) nucleic acid sequence encoding chalcone synthase activity, wherein chalcone synthase forms naringenin chalcone from malonyl-CoA and p-coumaroyl-CoA; (vi) nucleic acid sequence encoding chalcone isomerase activity, wherein chalcone isomerase forms naringenin from naringenin chalcone; (vii) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (viii) any combinations thereof. In certain embodiments, the flavonoid is catechin.
In yet another aspect, the invention provides a method of increasing the production of flavonoids or anthocyanins, the method comprising: providing an engineered host cell that comprises one or more genetic modifications resulting in production of flavonoid or anthocyanin from a carbon source that can also be an energy source, through multiple chemical intermediates, by the engineered host cell. In certain embodiments, the production of flavonoid or anthocyanin from a carbon source that can also be an energy source occurs through enzymatic transformation.
In certain embodiments, the carbon source is selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol; and (vi) any combination thereof In certain embodiments, the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol; and (vi) any combination thereof. In certain embodiments, one or more genetic modifications lead to increase metabolic flux to flavonoid precursors or cofactors. In certain embodiments, one or more genetic modifications cause increased metabolic flux to flavonoid precursors. In certain embodiments, one or more genetic modifications cause reduction in the formation of byproducts. In certain embodiments, one or more genetic modifications are selected from: (i) one or more modifications for over-expressing one or more endogenous genes in the engineered host cells;
(ii) one or more modifications for under-expressing one or more endogenous genes in the engineered host cells; (iii) one or more genetic modification is expressing one or more non-native genes in the engineered host cells; and (iv) a combination thereof. In certain embodiments, the engineered host cell is cultured in a medium comprising molecules selected from: tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid, wherein one or more of the selected molecules are the chemical intermediates, including molecules in the biosynthesis pathway or cofactors. In certain embodiments, the engineered host cell comprises at least one or more nucleic acid sequences selected from: (i) a nucleic acid sequences encoding tyrosine ammonia lyase activity; (ii) a nucleic acid sequences encoding phenylalanine ammonia lyase activity; (iii) cinnamate 4-hydroxylase; and (iv) any combination thereof In certain embodiments, the engineered host cell comprises at least one or more peptides selected from:
(i) chalcone isomerase; (ii) chalcone synthase; (iii) a fusion protein comprises a chalcone synthase and a chalcone isomerase; and (iv) any combination thereof. In certain embodiments, the engineered cell is E. Coil. In certain embodiments, one or more genetic modifications decreases fatty acid biosynthesis. In certain embodiments, the engineered host cell comprises an exogenous nucleic acid sequence selected from: (i) nucleic acid sequences encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (v) any combinations thereof In certain embodiments, the engineered host cell comprises an exogenous nucleic acid sequence selected from the group consisting of: (i) nucleic acid sequence encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acid sequence encoding 4-courmarate-CoA ligase activity, wherein 4-courmarate-CoA ligase forms p-coumaroyl-CoA from coumaric acid (v) nucleic acid sequence encoding chalcone synthase activity, wherein chalcone synthase forms naringenin chalcone from malonyl-CoA and p-coumaroyl-CoA; (vi) nucleic acid sequence encoding chalcone isomerase activity, wherein chalcone isomerase forms naringenin from naringenin chalcone; (vii) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (viii) any combinations thereof. In certain embodiments, the flavonoid is catechin.
In yet another aspect, the invention provides a plurality of engineered host cells, wherein each of the plurality of the engineered host cells comprises one or more genetic modifications resulting in production of flavonoid or anthocyanin from a carbon source that can also be an energy source, through multiple chemical intermediates. In certain embodiments, the production of flavonoid or anthocyanin from a carbon source that can also be an energy source occurs through enzymatic transformation. In certain embodiments, the carbon source is selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol; and (vi) any combination thereof. In certain embodiments, the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol; and (vi) any combination thereof In certain embodiments, one or more genetic modifications lead to increase metabolic flux to flavonoid precursors or cofactors. In certain embodiments, one or more genetic modifications lead to increase metabolic flux to flavonoid precursors or cofactors. In certain embodiments, one or more genetic modifications cause reduction of formation of byproducts. In certain embodiments, one or more genetic modifications are selected from: (i) one or more modifications for over-expressing one or more endogenous genes in the engineered host cells; (ii) one or more modifications for under-expressing one or more endogenous genes in the engineered host cells; (iii) one or more genetic modification is expressing one or more non-native genes in the engineered host cells; and (iv) a combination thereof. In certain embodiments, at least one of the engineered cells from the plurality of the engineered host cells is cultured in a medium comprising molecules selected from: tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid, wherein one or more of the selected molecules are the chemical intermediates, including molecules in biosynthesis pathway or cofactors. In certain embodiments, at least one of the engineered cells from the plurality of the engineered host cells comprise at least one or more nucleic acid sequences selected from: (i) nucleic acid sequences encoding tyrosine ammonia lyase activity; (ii) nucleic acid sequences encoding phenylalanine ammonia lyase activity; (iii) nucleic acid sequences encoding cinnamate 4-hydroxylase activity; (iv) nucleic acid sequences encoding 4-courmarate-CoA
ligase (4CL) activity; and (v) any combination thereof. In certain embodiments, at least one of the engineered host cell from the plurality of engineered host cells comprise at least one or more peptides selected from: (i) chalcone isomerase; (ii) chalcone synthase; (iii) a fusion protein comprises a chalcone synthase and a chalcone isomerase; and (iv) any combination thereof.
In certain embodiments, at least one the engineered host cell is E. coil. In certain embodiments, one or more genetic modifications decreases fatty acid biosynthesis. In certain embodiments, at least one of the engineered host cell from the plurality of the engineered host cells comprises an exogenous nucleic acid sequence selected from: (i) nucleic acid sequence encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (v) any combinations thereof. In certain embodiments, the engineered host cell comprises an exogenous nucleic acid sequence selected from the group consisting of: (i) nucleic acid sequence encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acid sequence encoding 4-courmarate-CoA ligase activity, wherein 4-courmarate-CoA ligase forms p-coumaroyl-CoA from coumaric acid (v) nucleic acid sequence encoding chalcone synthase activity, wherein chalcone synthase forms naringenin chalcone from malonyl-CoA and p-coumaroyl-CoA; (vi) nucleic acid sequence encoding chalcone isomerase activity, wherein chalcone isomerase forms naringenin from naringenin chalcone; (vii) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (viii) any combinations thereof. In certain embodiments, the flavonoid is catechin.
In yet another aspect, the invention provides a method of increasing the production of flavonoids or anthocyanins, the method comprising: providing a plurality of engineered host cells, wherein each of the plurality of the engineered host cell comprises one or more genetic modifications resulting production of flavonoid or anthocyanin from a carbon source that can also be an energy source, through multiple chemical intermediates, by the engineered host cell.
In certain embodiments, the production of flavonoid or anthocyanin from a carbon source that can also be an energy source occurs through enzymatic transformation. In certain embodiments, the carbon source is selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol; and (vi) any combination thereof. In certain embodiments, the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol; and (vi) any combination thereof In certain embodiments, one or more genetic modifications lead to increase metabolic flux to flavonoid precursors or cofactors. In certain embodiments, one or more genetic modifications lead to increase metabolic flux to flavonoid precursors or cofactors. In certain embodiments, one or more genetic modifications cause reduction of formation of byproducts. In certain embodiments, one or more genetic modifications are selected from: (i) one or more modifications for over-expressing one or more endogenous genes in the engineered host cells; (ii) one or more modifications for under-expressing one or more endogenous genes in the engineered host cells;
(iii) one or more genetic modification is expressing one or more non-native genes in the engineered host cells; and (iv) a combination thereof. In certain embodiments, at least one of the engineered cells from the plurality of the engineered host cells is cultured in a medium comprising molecules selected from: tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid, wherein one or more of the selected molecules are the chemical intermediates, including molecules in biosynthesis pathway or cofactors. In certain embodiments, at least one of the engineered cells from the plurality of the engineered host cells comprise at least one or more nucleic acid sequences selected from: (i) nucleic acid sequences encoding tyrosine ammonia lyase activity; (ii) nucleic acid sequences encoding phenylalanine ammonia lyase activity; (iii) nucleic acid sequences encoding cinnamate 4-hydroxylase activity;
.. (iv) nucleic acid sequences encoding 4-courmarate-CoA ligase (4CL) activity; and (v) any combination thereof. In certain embodiments, at least one of the engineered host cell from the plurality of engineered host cells comprise at least one or more peptides selected from: (i) chalcone isomerase; (ii) chalcone synthase; (iii) a fusion protein comprises a chalcone synthase and a chalcone isomerase; and (iv) any combination thereof. In certain embodiments, at least one the engineered host cell is E. coil. In certain embodiments, one or more genetic modifications decreases fatty acid biosynthesis. In certain embodiments, at least one of the engineered host cell from the plurality of the engineered host cells comprises an exogenous nucleic acid sequence selected from: (i) nucleic acid sequence encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate;
(ii) nucleic acid .. sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (v) any combinations thereof. In certain embodiments, the engineered host cell comprises an exogenous nucleic acid sequence selected from the group consisting of: (i) nucleic acid sequence encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acid sequence encoding 4-courmarate-CoA ligase activity, wherein 4-courmarate-CoA ligase forms p-coumaroyl-CoA from coumaric acid (v) nucleic acid sequence encoding chalcone synthase activity, wherein chalcone synthase forms naringenin chalcone from malonyl-CoA
and p-coumaroyl-CoA; (vi) nucleic acid sequence encoding chalcone isomerase activity, wherein chalcone isomerase forms naringenin from naringenin chalcone; (vii) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (viii) any combinations thereof In certain embodiments, the flavonoid is catechin.
In yet another aspect, the engineered host cell comprises one or more genetic modifications to increase the production and/or availability of malonyl-CoA.
In certain embodiments, the production and/or availability of malonyl-CoA is increased by transformation of acetyl-CoA to malonyl-CoA. In certain embodiments, the engineered host cell comprises one or more genetic modifications selected from: (i) expression of acetyl-CoA
carboxylase (ACC);
and (ii) overexpression of acetyl-CoA carboxylase. In another embodiment, the engineered host cell is an E. coil. In certain embodiments, the E. coil cell further comprises genes from fungi. In certain embodiments, the acetyl-CoA carboxylase is from: Mucor circinelloides, Rhodotorula toruloides, Lipomyces starkeyi, and Ustilago maydis, and orthologs of acetyl-CoA carboxylase having at least 50% amino acid identity to the acetyl-CoA carboxylase of these aforementioned species. In certain embodiments, one or more genetic modification is deletion or attenuation of one or more fatty biosynthetic genes resulting in decrease in fatty acid biosynthesis. In certain embodiments, one or more genetic modification is overexpression of acetyl-CoA
synthase (ACS). In certain embodiments, the acetyl-CoA synthase is selected from:
acetyl-CoA synthase gene of E. coil, acetyl-CoA synthase gene of Salmonella typhimurium, and orthologs of acetyl-CoA synthase gene in any other species having at least 50% amino acid identity to the acetyl-CoA synthase gene of E. coil and Salmonella typhimurium. In certain embodiments, one or more genetic modification is selected from a group consisting of: (i) overexpression a gene encoding pyruvate dehydrogenase (PDH), wherein the PDH may include E354K mutation; (ii) exogenous nucleic acid sequence encoding a malonyl-CoA synthetase; (iii) upregulation of endogenous pantothenate kinase (PanK), wherein PanK is not feedback inhibited by coenzyme A; (iv) exogenous nucleic acid sequence encoding a malonate transporter; and (v) any combinations thereof. In certain embodiments, the malonyl-CoA synthetase is selected from of malonyl-CoA
synthetases of Streptomyces coelicolor, Rhodopseudomonas palustris, or a malonyl-CoA
synthetase having at least 50% identity to any of these or other naturally occurring malonyl-CoA
synthetases. In certain embodiments, one or more genetic modifications to decrease fatty acid biosynthesis is selected from: (i) mutation or downregulation of a gene encoding malonyl-CoA-ACP transacylase (E. coil fabD); (ii) modifications to the gene beta-ketoacyl-ACP synthase II (E.
coil fabF); (iii) downregulation of beta-ketoacyl-ACP synthase I enzyme (E.
coil fabB); (iv) downregulation of acyl carrier protein (E. coil acpP); and (v) any combinations thereof. In certain embodiments, the engineered host cell comprises peptides selected from: (i) acetyl-CoA
carboxylase (ACC) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 15 or SEQ ID NO: 16; (ii) malonate CoA-transferase having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 19;
(iii) acetyl-CoA
synthase (ACS) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 16; (iv) malonyl-CoA synthase having an amino acid sequence at least 80%
identical SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 79; (v) malonate transporter having an amino acid sequence at least 80% identical to SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO:
82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, or SEQ ID NO:
87; (vi) pantothenate kinase having an amino acid sequence at least 80% identical to SEQ ID NO: 88, SEQ ID NO: 89, or SEQ ID NO: 90; and (vii) any combinations thereof.
In another aspect, the invention provides a method of increasing the production of flavonoids comprising an engineered host cell, wherein the one or more engineered host cells comprise one or more genetic modifications to increase the production and/or availability of malonyl-CoA. In certain embodiments, the production and/or availability of malonyl-CoA is increased by transformation of acetyl-CoA to malonyl-CoA. In certain embodiments, the engineered host cell comprises one or more genetic modifications selected from: (i) expression of acetyl-CoA carboxylase (ACC); and (ii) overexpression of acetyl-CoA
carboxylase. In another embodiment, the engineered host cell is an E. coil. In certain embodiments, the E. coil cell further comprises genes from fungi. In certain embodiments, the acetyl-CoA
carboxylase is from: Mucor circinelloides, Rhodotorula toruloides, Lipomyces starkeyi, and Ustilago maydis, and orthologs of acetyl-CoA carboxylase having at least 50% amino acid identity to the acetyl-CoA carboxylase of these aforementioned species. In certain embodiments, one or more genetic modification is deletion or attenuation of one or more fatty biosynthetic genes resulting in decrease in fatty acid biosynthesis. In certain embodiments, one or more genetic modification is overexpression of acetyl-CoA synthase (ACS). In certain embodiments, the acetyl-CoA synthase is selected from: acetyl-CoA synthase gene of E. coil, acetyl-CoA synthase gene of Salmonella typhimurium, and orthologs of acetyl-CoA synthase gene in any other species having at least 50% amino acid identity to the acetyl-CoA synthase gene of E. coil and Salmonella typhimurium.
In certain embodiments, one or more genetic modification is selected from a group consisting of:
(i) overexpression a gene encoding pyruvate dehydrogenase (PDH), wherein the PDH may include E354K mutation; (ii) exogenous nucleic acid sequence encoding a malonyl-CoA
synthetase; (iii) upregulation of endogenous pantothenate kinase (PanK), wherein PanK is not feedback inhibited by coenzyme A; (iv) exogenous nucleic acid sequence encoding a malonate transporter; and (v) any combinations thereof. In certain embodiments, the malonyl-CoA
synthetase is selected from of malonyl-CoA synthetases of Streptomyces coelicolor, Rhodopseudomonas palustris, or a malonyl-CoA synthetase having at least 50%
identity to any of these or other naturally occurring malonyl-CoA synthetases. In certain embodiments, one or more genetic modifications to decrease fatty acid biosynthesis is selected from: (i) mutation or downregulation of a gene encoding malonyl-CoA-ACP transacylase (E. coil fabD);
(ii) modifications to the gene beta-ketoacyl-ACP synthase II (E. coil fabF); (iii) downregulation of beta-ketoacyl-ACP synthase I enzyme (E. coil fabB); (iv) downregulation of acyl carrier protein (E. coil acpP); and (v) any combinations thereof. In certain embodiments, the engineered host cell comprises peptides selected from: (i) acetyl-CoA carboxylase (ACC) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 15 or SEQ ID NO:
16; (ii) malonate CoA-transferase having an amino acid sequence at least 80%
identical to the polypeptide set forth in SEQ ID NO: 19; (iii) acetyl-CoA synthase (ACS) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 16;
(iv) malonyl-CoA
synthase having an amino acid sequence at least 80% identical SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 79; (v) malonate transporter having an amino acid sequence at least 80%

identical to SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ
ID NO:
84, SEQ ID NO: 85, SEQ ID NO: 86, or SEQ ID NO: 87; (vi) pantothenate kinase having an amino acid sequence at least 80% identical to SEQ ID NO: 88, SEQ ID NO: 89, or SEQ ID NO:
90; and (vii) any combinations thereof.
In another aspect, the invention provides an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase endogenous biosynthesis of tyrosine. In certain embodiments, one or more genetic modifications comprises upregulation of 3-deoxy-D-arabino-heptulosonate synthase. In certain embodiments, one or more genetic modifications are selected from: (i) upregulation of chorismate mutase; (ii) upregulation of prephenate dehydrogenase; (iii) overexpression of shikimate kinase; (iv) overexpression of shikimate dehydrogenase; and (v) any combinations thereof. In certain embodiments, one or more genetic modifications comprises downregulation of L-phenylalanine biosynthetic pathway.
In certain embodiments, one or more genetic modifications comprises expression of exogenous phosphoenolpyruvate synthase (ppsA). In certain embodiments, one or more genetic modifications comprises expression of exogenous transketolase (tktA). In certain embodiments, wherein the one or more genetic modifications comprises disruption of tyrR
gene. In certain embodiments, one or more genetic modifications are selected from a group consisting of: (i) expression or overexpression of (D146N) variant of phospho-2-dehydro-3-deoxyheptonate aldolase; (ii) expression or overexpression of variant of 3-dehydroquinate synthase (aroB); (iii) overexpression of transketolase tktA; (iv) deletion of shikimate kinase (aroK); (v) deletion of tyrR; (vi) expression or overexpression of A3 54V variant of chorismate mutase (tyrA); (vi) and any combination thereof.
In another aspect, the invention provides a method of increasing endogenous biosynthesis of tyrosine comprising an engineered cell, wherein the engineered host cell comprises one or more genetic modifications to increase endogenous biosynthesis of tyrosine. In certain embodiments, one or more genetic modifications comprises upregulation of 3-deoxy-D-arabino-heptulosonate synthase. In certain embodiments, one or more genetic modifications are selected from: (i) upregulation of chorismate mutase; (ii) upregulation of prephenate dehydrogenase; (iii) overexpression of shikimate kinase; (iv) overexpression of shikimate dehydrogenase; and (v) any combinations thereof In certain embodiments, one or more genetic modifications comprises downregulation of L-phenylalanine biosynthetic pathway. In certain embodiments, one or more genetic modifications comprises expression of exogenous phosphoenolpyruvate synthase (ppsA).
In certain embodiments, one or more genetic modifications comprises expression of exogenous transketolase (tktA). In certain embodiments, wherein the one or more genetic modifications comprises disruption of tyrR gene. In certain embodiments, one or more genetic modifications are selected from a group consisting of: (i) expression or overexpression of (D146N) variant of phospho-2-dehydro-3-deoxyheptonate aldolase; (ii) expression or overexpression of variant of 3-dehydroquinate synthase (aroB); (iii) overexpression of transketolase tktA;
(iv) deletion of shikimate kinase (aroK); (v) deletion of tyrR; (vi) expression or overexpression of A354V
variant of chorismate mutase (tyrA); (vi) and any combination thereof.
In another aspect, the invention provides an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase transformation of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G). In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase. In certain embodiments, the anthocyanin synthase is selected from: (i) anthocyanin synthase of Car/ca papaya (SEQ. ID NO:13); (ii) the anthocyanin synthase has an amino acid sequence at least 80%
identical to SEQ. ID NO: 66, SEQ. ID NO: 67, SEQ. ID NO: 68, or SEQ. ID NO:
69; (iii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ.
ID NO: 13; and (iv) any combinations thereof In certain embodiments, one or more engineered host cells comprises flavonoid-3-glucosyl transferase (3GT). In certain embodiments, flavonoid-3-glucosyl transferase is selected from: (i) flavonoid-3-glucosyl transferase in Vitis labrusca (SEQ. ID
NO:14); (ii) the flavonoid-3-glucosyl transferase has an amino acid sequence at least 80%
identical to SEQ. ID NO: 70, SEQ. ID NO: 71, SEQ. ID NO: 72, or SEQ. ID NO:
73; and (iii) any combinations thereof In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, the one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, the one or more genetic modifications are selected from a group consisting of: (i) anthocyanin synthase, (ii) flavonoid-3-glucosyl transferase (3GT), and (iii) a combination thereof.

In another aspect, the invention provides a method for increasing the production of flavonoids comprising an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase transformation of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G). In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase. In certain embodiments, the anthocyanin synthase is selected from: (i) anthocyanin synthase of Car/ca papaya (SEQ. ID NO:13); (ii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 66, SEQ. ID NO: 67, SEQ. ID NO: 68, or SEQ. ID NO: 69; (iii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 13; and (iv) any combinations thereof.
In certain embodiments, one or more engineered host cells comprises flavonoid-3-glucosyl transferase (3GT). In certain embodiments, flavonoid-3-glucosyl transferase is selected from: (i) flavonoid-3-glucosyl transferase in Vitis labrusca (SEQ. ID NO:14); (ii) the flavonoid-3-glucosyl transferase has an amino acid sequence at least 80% identical to SEQ. ID NO:
70, SEQ. ID NO:
71, SEQ. ID NO: 72, or SEQ. ID NO: 73; and (iii) any combinations thereof. In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, the one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, the one or more genetic modifications are selected from a group consisting of: (i) anthocyanin synthase, (ii) flavonoid-3-glucosyl transferase (3GT), and (iii) a combination thereof.
In another aspect, the invention provides a method of increasing the transformation of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G), delphinidin or gallocatechin to delphindin-3-glucoside (De3G), or afzelechin or pelargonidin to pelargonidin-3-glucoside (Pe3G) comprising anthocyanin synthase, wherein the anthocyanin synthase is selected from: (i) anthocyanin synthase of Car/ca papaya (SEQ. ID NO:13); (ii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 66, SEQ. ID NO: 67, SEQ. ID NO:
68, or SEQ. ID NO: 69; (iii) the anthocyanin synthase has an amino acid sequence at least 80%
identical to SEQ. ID NO: 13; and (iv) any combinations thereof In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, the one or more genetic modifications are selected from a group consisting of: (i) anthocyanin synthase, (ii) flavonoid-3-glucosyl transferase (3GT), and (iii) a combination thereof.
In another aspect, the invention provides a method of increasing the transformation of cyanidin to cyanidin-3-glucoside (Cy3G), delphindin to delphindin-3-glucoside (De3G), or pelargonidin to pelagonidin-3-glucoside (Pe3G), comprising flavonoid-3-glucosyl transferase (3GT), wherein the flavonoid-3-glucosyl transferase is selected from: (i) flavonoid-3-glucosyl transferase in Vitis labrusca (SEQ. ID NO:14); (ii) the flavonoid-3-glucosyl transferase has an amino acid sequence at least 80% identical to SEQ. ID NO: 70, SEQ. ID NO: 71, SEQ. ID NO:
72, or SEQ. ID NO: 73; and (iii) any combinations thereof.
In another aspect, the invention provides an engineered host cell comprises one or more genetic modifications to increase the production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictoyl (EDL), and/or pentahydroxyflayaone (PHF), wherein the engineered host cell comprises cytochrome P450 reductase (CPR) and at least one of flavanone-3-hydroxylase (F3H), flavanone-3'-hydroxylase (F3'H), or flavonoid 3',5'-hydroxylase (F3'5'H). In certain embodiments, the precursor for increase in production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictoyl (EDL), and/or pentahydroxyflayanone (PHF) is naringenin and/or dihydrokaempferol (DHK). In certain embodiments, the engineered host cell further comprises peptides selected from a group consisting of: (i) flavonoid 3'-hydroxylase (F3'H); (ii) cytochrome P450 reductase (CPR); and (iii) any combination thereof In certain embodiments, the engineered host cell produces eriodictyol or taxifolin. In certain embodiments, the engineered host cell further comprises flavonoid 3',5'-hydroxylase (F3'5'H). In certain embodiments, the engineered host cell produces pentahydroxyflavone or dihydromyricetin. In certain embodiments, flavonoid 3'-hydroxylase (F3'H) is truncated to remove the N-terminal leader sequence. In certain embodiments, cytochrome P450 reductase (CPR) is truncated to remove the N-terminal leader sequence. In certain embodiments, flavonoid 3'-hydroxylase (F3'H) is fused with cytochrome P450 reductase (CPR). In certain embodiments, flavonoid 3',5'-hydroxylase (F3'5'H) is fused with cytochrome P450 reductase (CPR). In certain embodiments, flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 7. In certain embodiments, flavanone-3'-hydroxylase (F3'H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 8.
In certain embodiments, cytochrome P450 reductase (CPR) has an amino acid sequence at least 80%
identical to the polypeptide set forth in SEQ ID NO. 9. In certain embodiments, flavonoid 3',5'-hydroxylase (F3'5'H) has an amino acid sequence at least 80% identical to the polypeptides selected from a group consisting of: (i) SEQ ID NO. 10, (ii) SEQ ID NO. 56, and (iii) SEQ ID
.. NO. 57. In certain embodiments, the engineered host cell further comprises cytochrome b5. In certain embodiments, cytochrome b5 has an amino acid sequence at least 80%
identical to the polypeptide set forth in SEQ ID NO. 98. In certain embodiments, wherein the flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to the polypeptides selected from a group consisting of: (i) SEQ ID NO. 7, (ii) SEQ ID NO. 45, (iii) SEQ ID NO. 46, (iv) SEQ ID NO. 47, and (v) SEQ ID NO. 48.
In another aspect, the invention provides method of increasing the production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictoyl (EDL), and/or pentahydroxyflayaone (PHF) comprising an engineered host cell, wherein the engineered host cell comprises cytochrome P450 reductase (CPR) and at least one of flavanone-3-hydroxylase (F3H), flavanone-3'-hydroxylase (F3'H), or flavonoid 3',5'-hydroxylase (F3'5'H). In certain embodiments, the precursor for increase in production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictoyl (EDL), and/or pentahydroxyflayanone (PHF) is naringenin and/or dihydrokaempferol (DHK). In certain embodiments, the engineered host cell further comprises peptides selected from a group consisting of: (i) flavonoid 3'-hydroxylase (F3'H); (ii) .. cytochrome P450 reductase (CPR); and (iii) any combination thereof In certain embodiments, the engineered host cell produces eriodictyol or taxifolin. In certain embodiments, the engineered host cell further comprises flavonoid 3',5'-hydroxylase (F3'5'H). In certain embodiments, the engineered host cell produces pentahydroxyflavone or dihydromyricetin. In certain embodiments, flavonoid 3'-hydroxylase (F3'H) is truncated to remove the N-terminal leader sequence. In certain embodiments, cytochrome P450 reductase (CPR) is truncated to remove the N-terminal leader sequence. In certain embodiments, flavonoid 3'-hydroxylase (F3'H) is fused with cytochrome P450 reductase (CPR). In certain embodiments, flavonoid 3',5'-hydroxylase (F3'5'H) is fused with cytochrome P450 reductase (CPR). In certain embodiments, flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to the polypeptide set forth .. in SEQ ID NO. 7. In certain embodiments, flavanone-3'-hydroxylase (F3'H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 8.
In certain embodiments, cytochrome P450 reductase (CPR) has an amino acid sequence at least 80%
identical to the polypeptide set forth in SEQ ID NO. 9. In certain embodiments, flavonoid 3',5'-hydroxylase (F3'5'H) has an amino acid sequence at least 80% identical to the polypeptides selected from a group consisting of: (i) SEQ ID NO. 10, (ii) SEQ ID NO. 56, and (iii) SEQ ID
NO. 57. In certain embodiments, the engineered host cell further comprises cytochrome 13,5. In certain embodiments, cytochrome b5 has an amino acid sequence at least 80%
identical to the polypeptide set forth in SEQ ID NO. 98. In certain embodiments, wherein the flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to the polypeptides selected from a group consisting of: (i) SEQ ID NO. 7, (ii) SEQ ID NO. 45, (iii) SEQ ID NO. 46, (iv) SEQ ID NO. 47, and (v) SEQ ID NO. 48.
VI. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S) FIG. 1 shows the metabolic pathway of flavonoid and anthocyanin bioproduction in engineered cells and methods of preparing anthocyanins described herein.
FIG. 2 shows structures of the flavonoid and anthocyanin molecules that may be produced using engineered cells and methods of preparing anthocyanins described herein.
FIG. 3 shows HPLC spectra showing peaks corresponding to the molecules prepared using engineered cells and methods of preparing anthocyanins described herein.
FIG. 4 shows the pathway of flavonoid and anthocyanin bioproduction in engineered cells and methods of preparing anthocyanins described herein.
VII. DETAILED DESCRIPTION OF THE INVENTION
The present application provides engineered cells for producing one or more flavonoids, cultures that include the engineered cells, and methods of producing one or more flavonoids, or at least one anthocyanin. The terms "flavonoid", "flavonoid product", or "flavonoid compound"
are used herein to refer to a member of a diverse group of phytonutrients found in almost all fruits and vegetables. As used herein, the terms "flavonoid", "flavonoid product", or "flavonoid compound" are used interchangeably to refer a molecule containing the general structure of a 15-carbon skeleton, which consists of two phenyl rings (A and B) and a heterocyclic ring.
Flavonoids may include, but are not limited to, isoflavone type (e.g., genistein), flavone type (e.g., apigenin), flavonol type (e.g., kaempferol), flavanone type (e.g., naringenin), chalcone type (e.g., phloretin), anthocyanidin type (e.g., cyanidin), catechins, flavanones, and flavanonols.
Flavonoid compounds of interest include, without limitation, naringenin, naringenin chalcone, eriodictyol, taxifolin, dihydrokaempferol, dihydroquercetin, dihydromyricetin, leucocyanidin, leucopelargonidin, leucodelphindin, pentahydroxyflavone, cyanidin, catechin, delphinidin, pelargonidin, and kaempferol. Anthocyanins are in the forms of anthocyanidin glycosides and acylated anthocyanins. Anthocyanin compounds of interest include, without limitation, cyanidin glycoside, delphinidin glycoside, pelargonidin glycoside, peonidin glycoside, and petunidin glycoside.
The terms 'precursor' or `flavonoid precursor' as used herein may refer to any intermediate present in the biosynthetic pathway that leads to the production of catechins or anthocyanins. flavonoid precursors may include, but are not limited to tyrosine, phenylalanine, coumaric acid, p-coumaroyl-CoA, malonyl-CoA, pyruvate, acetyl-CoA, and naringenin.
Cells engineered for the production of a flavonoid or an anthocyanin can have one or multiple modifications, including, without limitation, the downregulation, disruption, or deletion of endogenous genes, the upregulation of an endogenous gene, and the introduction of exogenous genes.
The term "non-naturally occurring", when used in reference to an enzyme is intended to mean that nucleic acids or polypeptides include at least one genetic alteration not normally found in a naturally occurring polypeptide or nucleic acid sequence. Naturally occurring nucleic acids, and polypeptides can be referred to as "wild-type" or "original". A host cell, organism, or microorganism that includes at least one genetic modification generated by human intervention can also be referred to as "non-naturally occurring", "engineered", "genetically engineered," or "recombinant".
A host cell, organism, or microorganism engineered to express or overexpress a gene or nucleic acid sequence, or to overexpress an enzyme or polypeptide has been genetically engineered through recombinant DNA technology to include a gene or nucleic acid sequence that does not naturally encode the enzyme or polypeptide or to express an endogenous gene at a level that exceeds its level of expression in a non-altered cell. As nonlimiting examples, a host cell, organism, or microorganism engineered to express or overexpress a gene or a nucleic acid sequence, or to overexpress an enzyme or polypeptide can have any modifications that affect a coding sequence of a gene, the position of a gene on a chromosome or regulatory elements associated with a gene. Overexpression of a gene can also be by increasing the copy number of a gene in the cell or organism. Similarly, a host cell, organism, or microorganism engineered to under-express or to have reduced expression of a gene, nucleic acid sequence, or to under-express an enzyme or polypeptide can have any modifications that affect a coding sequence of a gene, the position of a gene on a chromosome or regulatory elements associated with a gene.
Specifically included are gene disruptions, which include any insertions, deletions, or sequence mutations into or of the gene or a portion of the gene that affect its expression or the activity of the encoded polypeptide. Gene disruptions include "knockout" mutations that eliminate expression of the gene. Modifications to under-express a gene also include modifications to regulatory regions of the gene that can reduce its expression.
The term "exogenous" or "heterologous" is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism. The molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material that may be introduced on a vehicle such as a plasmid. Therefore, the term "endogenous" refers to a referenced molecule or activity that is naturally present in the host.
Genes or nucleic acid sequences can be introduced stably or transiently into a host cell using techniques well known in the art including, but not limited to, conjugation, electroporation, chemical transformation, transduction, and transfection. Optionally, for exogenous expression in E. coil or other prokaryotic cells, some nucleic acid sequences in the genes or cDNAs of eukaryotic nucleic acids can encode targeting signals such as an N-terminal mitochondrial or other targeting signal, which can be removed before transformation into prokaryotic host cells, if desired. Furthermore, genes can be subjected to codon optimization with techniques well known in the art to achieve optimized expression of the proteins.
The percent identity (% identity) between two sequences is determined when sequences are aligned for maximum homology. Algorithms well known to those skilled in the art, such as Align, BLAST, Clustal Omega, and others compare and determine a raw sequence similarity or identity, and also determine the presence or significance of gaps in the sequence which can be assigned a weight or score. Such algorithms also are known in the art and are similarly applicable for determining nucleotide or amino acid sequence similarity or identity and can be useful in identifying orthologs of genes of interest. Additional sequences added to a polypeptide sequence, such as but not limited to immunodetection tags, purification tags, localization sequences (presence or absence), etc., do not affect the % identity.
A homolog is a gene or genes that have the same or identical functions in different organisms. Genes that are orthologous can encode proteins with sequence similarity of about 45% to 100% amino acid sequence identity, and more preferably about 60% to 100% amino acid sequence identity. Genes can also be considered orthologs if they share three-dimensional structure but not necessarily sequence similarity, of a sufficient amount to indicate that they have evolved from a common ancestor to the extent that the primary sequence similarity is not identifiable. Paralogs are genes related by duplication within a genome, and can evolve new functions, even if these are related to the original one.
An engineered cell for producing flavonoids include an exogenous nucleic acid sequence encoding tyrosine ammonia lyase (TAL) activity (alternatively or in addition, an exogenous nucleic acid encoding phenylalanine ammonia-lyase (PAL) activity and an exogenous nucleic acid encoding cinnamate-4-hydroxylase (C4H) activity), an exogenous nucleic acid sequence encoding 4-coumarate-CoA ligase (4CL) activity, an exogenous nucleic acid sequence encoding chalcone synthase (CHS) activity, and an exogenous nucleic acid sequence encoding chalcone isomerase (CHI) activity. Optionally, the engineered cell can further include an exogenous nucleic acid sequence encoding an exogenous nucleic acid sequence encoding flavanone-3-hydroxylase (F3H) activity, an exogenous nucleic acid sequence encoding flavonoid 3'-hydroxlase (F3'H) activity or flavonoid 3',5'-hydroxylase (F3'5'H), an exogenous nucleic acid sequence encoding cytochrome P450 reductase (CPR) activity, an exogenous nucleic acid sequence encoding dihydroflavono1-4-reductase (DFR) activity, and/or an exogenous nucleic acid sequence encoding leucoanthocyanidin reductase (LAR) activity.
Tyrosine ammonia-lyase (TAL) can be, for example, a member of the aromatic amino acid deaminase family that catalyzes the elimination of ammonia from L-tyrosine to yield p-coumaric acid. An exemplary tyrosine ammonia lyase is the Saccharothrix espanaensis tyrosine ammonia lyase (TAL; SEQ ID NO: 1). Also considered for use in the engineered cells provided herein are TALs with SEQ ID NOS: 23-26, TALs listed in Table 1, TAL homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID:1 that have the activity of a tyrosine ammonia lyase that produces p-coumaric acid from tyrosine.
Table 1. Tyrosine ammonia-lyase Organism GenBank Accession Number Rhodotorula glutini AGZ04575.1 Flavobacterium johnsoniae WPO12023194.1 Herpetosiphon aurantiacus ABX02653.1 Rhodobacter capsulatus ADE83766.1 Saccharothrix espanaensis AKE50820.1 Trichosporon cutaneum AKE50834.1 Similar to tyrosine ammonia-lyase, phenylalanine ammonia-lyase (PAL) can be a member of the aromatic amino acid deaminase family that catalyzes the non-oxidative deamination of L-phenylalanine to form trans-cinnamic acid. An exemplary phenylalanine ammonia-lyase is the Brevi bacillus laterosporus phenylalanine ammonia-lyase (PAL; SEQ ID
NO :2). Also considered for use in the engineered cells provided herein are PALs with SEQ ID
NOS: 27-29, PAL homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2 that have the activity of a phenylalanine ammonia lyase that produces trans-cinnamic acid from phenylalanine.
Cinnamate-4-hydroxylase (C4H) belongs to the cytochrome P450-dependent monooxygenase family and catalyzes the formation of p-coumaric acid from trans-cinnamic acid.
Considered for use in the engineered cells provided herein are C4H of Helianthus annuus L.
(C4H; SEQ ID NO: 3), C4Hs with SEQ ID NOS: 30-32, and C4H homologs of other species, as well as variants of naturally occurring C4Hs having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to the SEQ ID NO: 3 (C4H, Helianthus annuus L.) that have the activity of a C4H.
4-coumarate-CoA ligase (4CL) catalyzes the activation of 4-coumarate to its CoA ester.
Considered for use in the engineered cells provided herein are 4CLs of Petroselinum crispum (SEQ ID NO: 4), 4CLs in Table 2, 4CLs with SEQ ID NOS: 33-36, and 4CL homologs of other species, as well as variants of naturally occurring 4CLs having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to SEQ ID No:
4 (4CL, Petroselinum crispum) that have the activity of a 4CL.
Table 2. 4-coumarate-CoA ligases Organism GenBank Accession Number Petroselinum crispum CAA31697.1 Camellia sinensis ASU87409.1 Capsicum annuum KAF3620173.1 Castanea mollissima KAF3954751.1 Daucus carota AIT52344.1 Gynura bicolor BAJ17664.1 Ipomoea purpurea AHJ60263.1 Lonicera japonica AGE10594.1 Lycium chinense QDL52638.1 Nelumbo nucifera XP 010265453.1 Nyssa sinensis KAA8540582.1 Solanum lycopersicum NP 001333770.1 Striga as/at/ca GER48539.1 The chalcone synthase (CHS) can be, for example, a type III polyketide synthase that sequentially condenses three molecules of malonyl-CoA with one molecule of p-coumaryol-CoA
to produce the naringenin precursor naringenin chalcone or naringenin. An exemplary chalcone synthase is the chalcone synthase of Petunia x hybrida (CHS, SEQ ID NO: 5).
Also considered for use in the engineered cells provided herein are the genes listed in Table 3, CHSs with SEQ
ID: 37-40, and CHS homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to SEQ ID NO: 5 (CHS, Petunia x hybrida) that have the activity of a chalcone synthase.
Table 3. Chalcone synthases Organism GenBank Accession Number Petunia hybrida AAF60297.1 Acer palmatum AWN08245.1 Callistephus chinensis CAA91930.1 Camellia japonica BAI66465.1 Capsicum annuum XP 016566084.1 Coffea arabica )CP 027118978.1 Curcuma alismatifolia ADP08987.1 Dendrobium catenatum ALE71934.1 Garcinia mangostana ACM62742.1 Iochroma calycinum AIY22758.1 Iris germanica BAE53636.1 Lilium speciosum BAE79201.1 Lonicera caerulea ALU09326.1 Lycium ruthenicum ATB56297.1 Magnolia lihiflora AHJ60259.1 Matthiola incana BBM96372.1 Morus alba var. multicaulis AHL83549.1 Nelumbo nucifera NP 001305084.1 Nyssa sinensis KAA8548459.1 Paeonia lactiflora AEK70334.1 Panax notoginseng QKV26463.1 Ranunculus asiaticus AYV99476.1 Rosa chinensis AEC13058.1 Theobroma cacao XP 007032052.2 Chalcone isomerase (CHI, also referred to as chalcone flavonone isomerase) catalyzes the stereospecific and intramolecular isomerization of naringenin chalcone into its corresponding (2S)-flavanones. Considered for use in the engineered cells provided herein are CHI of Medicago sativa (SEQ ID NO: 6), CHI of Table 4, CHIs with SEQ ID NOS: 41-44, and CHI
homologs of other species, as well as variants of naturally occurring CHI having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to SEQ ID
NO: 6 (CHI, Medicago sativa) that have the activity of a chalcone isomerase.
Table 4. Chalcone Isomerases Organism GenBank Accession Number Medicago sativa AGZ04578.1 Dendrobium hybrid cultivar AGY46120.1 Abrus precatorius XP 027366189.1 Antirrhinum majus BA032070.1 Arachis duranensis XP 015942246.1 Astragalus membranaceus ATY39974.1 Camellia sinensis XP 028119616.1 Castanea mollissima KAF3958409.1 Cephalotus follicularis GAV77263.1 Clarkia gracilis subsp.
QPF47150.1 sonomensis Dianthus caryophyllus CAA91931.1 Glycyrrhiza uralensis AX059749.1 Handroanthus impetiginosus PIN05040.1 Lotus japonicus CAD69022.1 Morus alba AFM29131.1 Phaseolus vulgaris XP 007142690.1 Pun/ca granatum ANB66204.1 Rhodamnia argentea XP 030524476.1 Spatholobus suberectus TKY50621.1 Trifolium subterraneum GAU12132.1 A nucleic acid sequence encoding a CHI can in some embodiments be fused to a nucleic acid sequence encoding a CHS in an engineered cell as provided herein, such that the CHI

activity is fused to the chalcone synthase activity, i.e., a fusion protein is produced in the engineered cell that has both condensing and cyclization activities.
Flavanone 3-hydroxylase (F3H) catalyzes the stereospecific hydroxylation of (2S)-naringenin to form (2R,3R)-dihydrokaempferol. Other substrates include (2S)-eriodictyol, (2S)-dihydrotricetin and (2S)-pinocembrin. Some F3H enzymes are bifunctional and also catalyzes as flavonol synthase (EC: 1.14.20.6). Considered for use in the engineered cells provided herein are F3H of Rubus occidentalis (SEQ ID NO: 7), F3Hs with SEQ ID NOS: 45-48, F3Hs listed in Table 5, and other F3H homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to SEQ ID NO:7 (F3H, Rubus occidentalis) that have the activity of a F3H.
Table 5. Flavanone 3-hydroxylases Organism GenBank Accession Number Rubus occidentalis ACM17897.1 Abrus precatorius XP 027347564.1 Nyssa sinensis KAA8547483.1 Camellia sinensis AAT68774.1 Morella rubra KAB1219056.1 Rosa chinensis PRQ47414.1 Malus domestica AAD26206.1 Vitis amurensis ALB75302.1 Iochroma elhpticum AMQ48669.1 Hibiscus sabdariffa ALB35017 Cephalotus follicularis GAV71832 Flavonoid 3'-hydroxylases (F3'H) belongs to the cytochrome P450 family with systematic name of flavonoid, NADPH:oxygen oxidoreductase (3'-hydroxylating).
In the flavonoid biosynthetic pathway, F3'H converts dihydrokaempferol to dihydroquercetin (taxifolin) or naringenin to eriodictyol. Considered for use in the engineered cells provided herein are F3'H of Brass/ca napus (F3'H; SEQ ID NO: 8), F3'H with SEQ ID NOS:
49-52, those listed in Table 6, and homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to these F3'H.
F3'H is a cytochrome P450 enzyme that requires a cytochrome P450 reductase (CPR) to function. Cytochrome P450 reductases are diflavin oxidoreductases that supply electrons to F3'Hs. The P450 reductase can be from the same species as F3'H or different species from F3'H.
Considered for use in the engineered cells provided herein are CPR of Catharanthus roseus (SEQ ID NO: 9), additional CPRs listed in Table 7, CPRs with SEQ ID NOS: 53-55, CPR
homologs of other species, and variants of naturally occurring CPRs having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%,at least 98%, or at least 99%
amino acid identity to these CPRs that have the activity of a CPR. In various embodiments, the N-terminal nucleic acid sequences in the genes of F3'H and/or CPR originated from eukaryotic cells can encode targeting leader peptides, which can be removed before introduction into prokaryotic host cells, if desired. In some embodiments, the hydroxylase complex HpaBC from E. coli was used to hydroxylate naringenin to eriodictyol or dihydrokaempferol to dihydroquercetin (taxifolin).
Table 6. Flavonoid 3'-hydroxylases Organism GenBank Accession Number Brass/ca napus ABC58722.1 Gerbera hybrid cultivar DI ABA64468.1 Cephalotus folhcularis GAV84063.1 Theobroma cacao XP 007037548.1 Phoenix dactyhfera XP 008791304.2 Table 7. Cytochrome P450 reductases Organism GenBank Accession Number Catharanthus roseus CAA49446.1 Brass/ca napus XP 013706600.1 Cephalotus follicularis GAV59576.1 Camellia sinensis XP 028084858.1 A nucleic acid sequence encoding a F3'H can in some embodiments be fused to a nucleic acid sequence encoding a CPR in an engineered cell as provided herein, such that the F3 'H
activity is fused to the CPR activity.
In the cells engineered to produce dihydomyricetin, flavonoid 3', 5'-hydroxylase (F3'5'H) can be used to convert dihydrokaempferol to dihydromyricetin or naringenin to pentahydroxyflavone, which is further converted to dihydromyricetin by a F3H.
F3'5'H has the systematic name flavanone,NADPH:oxygen oxidoreductase and catalyzes the formation of 3',5'-dihydroxyflavanone from flavanone. An exemplary F3'5'H is the Delphinium grandiflorum F3'5'H (SEQ ID NO: 10), Also considered for use in the engineered cells provided herein include F3'5'H with SEQ ID NOS:56-57, F3'5'H homologs of other species, and variants of naturally occurring F3'5'H having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to SEQ ID NOS:10 that have the activity of a F3'5'H.
Dihydroflavonol 4-reductase (DFR) acts on (+) ¨ dihydrokaempferol (DHK), (+)-dihydroquercetin (Taxifolin, DHQ), or dihydromyricein (DHM) to reduce those compounds to the corresponding cis-flavan-3,4-diol (DHK to leucopelargonidin; Taxifolin to leucocyanidin;
DHM to leucodelphinidin). An exemplary DFR is the Anthurium andraeanum DFR
(SEQ ID
NO: 11). Also considered for use in the engineered cells provided herein include DFRs in Table 8, DFRs with SEQ ID NOS: 58-61, and DFR homologs of other species, as well as variants of naturally occurring DFR having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to SEQ ID NO: 11.
Table 8. Dihydroflavonol 4-reductases Organism GenBank Accession Number Eustoma grandiflorum BAD34461.1 Anthurium andraeanum AAP20866.1 Camellia sinensis AAT66505.1 Morella rubra KAB1203810.1 Dendrobium moniliforme AEB96144.1 Fragaria x ananassa AHL46451.1 Rosa chinensis XP 024167119.1 Acer palmatum AWN08247.1 Nyssa sinensis KAA8531902.1 Vitis amurensis 182380.1 Abrus precatorius XP 027329642.1 Angelonia angustifolia AHM27144.1 Pyrus pyrifolia Q84KP0.1 Theobroma cacao XP 017985307 Theobroma cacao XP 007051597.2 Brassica oleracea var. capitata QK029328.1 Rubus idaeus AXK92786.1 Citrus sinensis AAY87035.1 Gerbera hybrida P51105.1 Cephalotus folhcularis GAV76940.1 Ginkgo biloba AGR34043.1 Dryopteris erythrosora QFQ61498.1 Dryopteris erythrosora QFQ61499.1 Cephalotus folhcularis GAV76942.1 Leucoanthocyanidin reductase (LAR) catalyzes the synthesis of catechin from 3,4-cis-leucocyanidin. LAR also synthesizes afzelechin and gallocatechin. Considered for use in the engineered cells provided herein are LAR of Desmodium uncinatum (SEQ ID NO:
12), LARs with SEQ ID NOS: 62-65, and LAR homologs of other species, as well as variants of naturally occurring LAR having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to SEQ ID NO: 12 (LAR, Desmodium uncinatum) that have the activity of a LAR.
Optionally, the cells are further engineered to include an anthocyanin synthase (ANS) which catalyzes the conversion of leucoanthocyanidin or catechin to anthocyanidin, leucopelargonidin to pelargonidin, or leucodelphinidin to delphinidin.
Considered for use in the engineered cells provided herein are ANS of Car/ca papaya (SEQ ID NO: 13), ANS
with SEQ
ID NOS: 66-69, and ANS homologs of other species, as well as variants of naturally occurring ANS having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to SEQ ID NO:13 (ANS, Car/ca papaya) that have the activity of a ANS.
Optionally, the cells are further engineered to include a flavonoid-3-glucosyl transferase (3GT) to generate anthocyanins by transfer of a sugar moiety such as, without limitation, UDP-a-D-glucose to anthocyanidins to form glycosylated anthocyanins. Considered for use in the engineered cells provided herein are 3GT of Vitis labrusca (SEQ ID NO:14), 3GT
with SEQ ID
NOS: 70-73, and 3GT homologs of other species, as well as variants of naturally occurring 3GT
having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to SEQ ID NO: 14 (3GT, Vitis labrusca) that have the activity of a 3GT.
In various aspects, host cells may be engineered for enhanced production of flavonoids or anthocyanins by introducing additional exogenous pathways and/or modifying endogenous metabolic pathways to remove or downregulate competitive pathways to reduce carbon loss, increase precursor supply, improve cofactor availability, reduce byproduct formation, or improve cell fitness. Enhancing or improving production of flavonoids or anthocyanins can be increasing yield, titer, or rate of production.
Thus, a host cell engineered for the production of a flavonoid or anthocyanin can be engineered to include any or any combination of: overexpression of an acetyl-CoA carboxylase (ACC) or an ACC variant; expression or overexpression of at least one enzyme for increasing cell's malonyl-CoA supply that does not rely on the ACC step; expression or overexpression of at least one enzyme to increase tyrosine supply; expression or overexpression of at least one enzyme to increase CoA availability for synthesizing precursors malonyl-CoA or p-coumaryol-CoA; expression or overexpression at least one enzyme to increase heme biosynthesis; deletion or downregulation of at least one fatty acid synthesis enzyme; at least one alcohol dehydrogenase, lactate dehydrogenase, pyruvate oxidase, phosphate acetyl transferase, or acetate kinase; at least one enzyme of a fatty acid degradation pathway, at least one thioesterase, or at least one TCA gene. The foregoing list of modifications is nonlimiting.
Malonyl-CoA is the direct precursor for chalcone synthase to perform sequential condensations with p-coumaryol-CoA. Malonyl-CoA supply can be increased by one or more modifications. Malonyl-CoA is synthesized by acetyl-CoA carboxylase (ACC) via the ATP-dependent carboxylation of acetyl-CoA in a multistep reaction. First, the biotin carboxylase domain catalyzes the ATP-dependent carboxylation of biotin using bicarbonate as a CO2 donor.
In the second reaction, the carboxyl-group is transferred from biotin to acetyl-CoA to form malonyl-CoA. In most eukaryotes, including fungi, both reactions are catalyzed by a large single chain protein, but in E. coil and other bacteria, the activity is catalyzed by a multi-subunit enzyme. Host cells can be engineered for example to express an exogenous acetyl-CoA
carboxylase or a variant ACC to increase malonyl-CoA synthesis from acetyl-CoA. For example, Mucor circinelloides (SEQ ID NO: 15) acetyl-CoA carboxylase can be introduced into the host cells. Additional examples of ACC genes that may be used in the engineered cells provided herein include, without limitation, the genes listed in Table 9, genes with SEQ ID NOS: 74-76, naturally occurring orthologs of these ACCs, or variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to referenced genes. Further, naturally occurring acetyl-CoA carboxylase genes can be further engineered to introduce single or multiple amino acid mutations to increase catalytic activity and/or remove feedback inhibition.
Table 9. Acetyl-CoA carboxylases Organism GenBank Accession Number Lipomyces starkeyi AJT60321.1 Rhodotorula toruloides GEM08739.1 Ustilago maydis XPO11390921.1 Mucor circinelloides EPB82652.1 Kalaharituber pfeihi KAF8466702.1 Aspergillus fumigatus KEY77072.1 Rhodotorula diobovata TNY18634.1 Leucosporidium creatinivorum ORY74050.1 Microbotryum intermedium SCV70467.1 Mixia osmundae GAA98306.1 Puccinia graminis KAA1079218.1 Suit/us occidentalis KAG1764021.1 Gymnopilus junonius KAF8909366.1 Additional strategies for increasing malonyl-CoA include increasing acetyl-CoA, which is converted to malonyl-CoA by acetyl-CoA carboxylase (ACC). Acetyl-CoA can be synthesized from acetate by an acyl-CoA ligase in an ATP-dependent reaction. Acetyl-CoA
synthetase (ACS) or acetate-CoA ligase (EC 6.2.1.1.) catalyzes the formation of a new chemical bond between acetate and CoA coenzyme A (CoA). ACSs with native activity on acetate will provide the function of increasing acetyl-CoA supply when cells are either supplied with acetate as a co-feed, or where acetate is produced as a by-product. Other acyl-CoA
ligases, having their main activity on other acid substrates, may also have substantial activity on acetate, and are viable candidates for providing acetate-CoA ligase activity in the engineered cells provided herein. The AC Ss expressed in the host cells can be prokaryotic or eukaryotic. Cultures of engineered host cells that overexpress a nucleic acid sequence encoding ACS
can optionally include acetate in the culture medium. Examples of acetyl-CoA synthase that can be expressed in a host cell engineered to produce a flavonoid or anthocyanin include, without limitation, the ACS
gene of E. coli, the ACS of Salmonella typhimurium (SEQ ID NO:16), and orthologs of these ACSs in other species having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to these ACSs.
Alternatively, or in addition, an engineered host cell can overexpress a gene encoding pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA, to increase acetyl-CoA
supply. PDH catalyzes an irreversible metabolic step, and the control of its activity is complex and involves control by its substrates and products. Nicotinamide adenine dinucleotide hydrogen (NADH), a product of the PDH reaction, is a competitive inhibitor of the PDH
complex. The NADH sensitivity of the PDH complex has been demonstrated to reside in LPD, the enzyme that interacts with NAD+ as a substrate. Thus, a variant of the Lpd subunit of PDH
can be expressed that includes one or more mutations that reduces inhibition of PDH by NADH.
Such an example is a LPD variant in E. coil that contains E354K mutation, and the mutated enzyme was less sensitive to NADH inhibition than the native LPD.
Alternatively, or in addition to strategies for increasing ACC activity and strategies for increasing acetyl-CoA, strategies for increasing malonyl-CoA by mechanisms that do not rely on the activity of an ACC can be employed. For example, a cell engineered to produce a flavonoid or an anthocyanin as provided herein can include an exogenous nucleic acid sequence encoding a malonyl-CoA synthetase (EC 6.2.1.14) that generates malonyl-CoA from malonate.
Acyl-CoA
synthetase catalyzes the conversion of a carboxylic acid to its acyl-CoA
thioester through an ATP-dependent two-step reaction. In the first step, the free fatty acid is converted to an acyl-AMP intermediate with the release of pyrophosphate. In the second step, the activated acyl group is coupled to the thiol group of CoA, releasing AMP and the acyl-CoA product.
Nonlimiting examples of malonyl-CoA synthetases include the malonyl-CoA synthetases of Streptomyces coelicolor (SEQ ID NO:17), matB of Rhodopseudomonas palustris (SEQ ID NO: 77), matB of Rhizobium sp, BUS003 (SEQ ID NO: 78), matB of Ochrobacrum sp. (SEQ ID NO:
79),or other homologs having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the referenced sequences. Malonate can optionally be added to the culture medium of a culture that includes a cell engineered to express a malonyl-CoA
synthetase. In Rhizobium trifohi, the math gene is part of the matABC operon, with matA encoding a malonyl-CoA decarboxylase and matC encoding a putative dicarboxylate carrier protein or malonate transporter. An engineered cell that includes an exogenous gene encoding a malonyl-CoA synthetase can also include an exogenous nucleic acid sequence encoding a malonate transporter, such as a malonate transporter encoded by a matC gene, for example of Streptomyces coelicolor (SEQ ID NO:18), of Rhizobiales bacterium (SEQ ID
NO:80), of Rhizobium leguminosarum (SEQ ID NO:81), of Agrobacterium vitis (SEQ
ID NO:
82), of Neorhizobium sp. (SEQ ID NO: 83), or a malonate transporter encoded by DctPQM of Sinorhizobium medicae, or encoding a malonyl-CoA transporter having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%
identity to a naturally-occurring malonate transporter. Cell cultures of a host cell engineered to express a malonyl-CoA synthetase and a malonate transporter can include a culture medium that includes malonate.
In additional embodiments, a cell engineered to produce a flavonoid or an anthocyanin is further engineered to include an exogenous nucleic acid sequence encoding malonate CoA-transferase (EC:2.8.3.3; also referred to as the alpha subunit of malonate decarboxylase) that makes malonyl-CoA by direct transfer of the CoA from acetyl-CoA. For example, the alpha subunit of malonate decarboxylase from the mdcACDE gene cluster in Acinetobacter calcoaceticus has the malonate CoA-transferase activity. The mdcA gene product, the a subunit, is malonate CoA-transferase, and mdcD gene product, the 0 subunit, is a malonyl-CoA
decarboxylase. The mdcE gene product, the y subunit, may play a role in subunit interaction to form a stable complex or as a codecarboxylase. The mdcC gene product, the 6 subunit, was an acyl-carrier protein, which has a unique CoA-like prosthetic group. When the a subunit is removed from the complex and incubated with malonate and acetyl-CoA, the acetyl-CoA moiety of the prosthetic group binds on an a subunit to exchange the acetyl group for a malonyl group.
As the thioester transfer should be thermodynamically favorable, the engineered cells can include a nucleic acid encoding a malonate CoA-transferase to increase malonyl-CoA supply.
Examples of mdcAs that can be expressed in an engineered cell as provided herein include, without limitation, mdcA of Acinetobacter calcoaceticus (SEQ ID NO: 19), mdcAs of Table 10, mdcAs with SEQ ID NOS: 84-87, or a transferase having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of these or other naturally occurring malonate CoA-transferases.
Table 10. Malonate CoA-transferases (malonate decarboxylase subunit alpha) Organism GenBank Accession Number Acinetobacter calcoaceticus AAB97627.1 Geobacillus sp. QNU36929.1 Acinetobacter johnsonii WP 087014029.1 Acinetobacter marinus WP 092618543.1 Acinetobacter rudis WPO16655668.1 Psychrobacter sp. G WP 020444454.1 Moraxella catarrhalis WP 064617969.1 Zoogloea sp. MBL0283742.1 Dechloromonas sp. KAB2923906.1 Stenotrophomonas rhizophila WP 123729366.1 Xanthomonas cucurbitae WP 159407614.1 In some embodiments, a cell engineered to produce flavonoids or anthocyanins is further engineered to increase the supply of coenzyme A (CoA) to increase its availability for producing acetyl-CoA, malonyl-CoA, and/or p-coumaroyl-CoA. Strategies for increasing CoA
supply include expressing or overexpressing at least one enzyme of a CoA biosynthesis pathway.
Pantothenate kinase (EC 2.7.1.33, PanK; CoaA) is the first enzyme in the coenzyme CoA
biosynthetic pathway. It phosphorylates pantothenate (vitamin B5) to form 4'-phosphopantothenate at the expense of a molecule of adenosine triphosphate (ATP). It is the rate-limiting step in the biosynthesis of CoA. Three distinct types of PanK
have been identified -PanK-I (found in bacteria), PanK-II (mainly found in eukaryotes, but also in the Staphylococci) and PanK-III, also known as CoaX (found in bacteria). In E. coil, pantothenate kinase is competitively inhibited by CoA itself, as well as by some CoA esters. The type III enzymes CoaX are not subject to feedback inhibition by CoA. In some embodiments, a host cell can be engineered to include a nucleic acid sequence encoding type III pantothenate kinase that is not feedback inhibited by coenzyme A, such as, without limitation, CoaX gene of Pseudomonas aeruginosa (EC:2.7.1.33, SEQ ID NO: 20), CoaX of Streptomyces sp. CL12509 (SEQ
ID NO:
88), CoaX of Streptomyces cinereus (SEQ ID: 89), or CoaX of Kitasatospora kifunensis (SEQ ID
NO: 90) Cultures of cells engineered for the production of flavonoids or anthocyanins can in some embodiments include a medium that includes pantothenate, a precursor of CoA
biosynthesis, and can optionally also include cysteine, used in the CoA
biosynthesis.

Additional strategies to increase malonyl-CoA flux to the flavonoid pathway include mutation or downregulation of one or more genes that function in fatty acid biosynthesis. Fatty acid biosynthesis directly competes with flavonoid biosynthesis for the precursor malonyl-CoA
and thus limits flavonoid formation. Without limiting the embodiments to any particular mechanism, limiting fatty acid biosynthesis can increase the malonyl-CoA
supply available for flavonoid biosynthesis. In some embodiments, the gene beta-ketoacyl-ACP
synthase II (E. coil fabF) can be disrupted, attenuated or deleted to reduce fatty acid biosynthesis. Another example of a fatty acid biosynthesis gene of a host cell that may be mutated or downregulated is a gene encoding malonyl-CoA-ACP transacylase (E. coil fabD). Other fatty acid biosynthesis genes of the engineered host cell that can be downregulated include a beta-ketoacyl-ACP
synthase I
enzyme (E. coil fabB) and/or acyl carrier protein (E. coil acpP).
Additional genetic modifications that may be present in a host cell engineered to produce flavonoids or anthocyanins include downregulation, disruption, or deletion of the gene targets that divert carbon flux to form byproducts such as ethanol, acetate, and lactate. They include genes encoding alcohol dehydrogenase, lactate dehydrogenase, pyruvate oxidase, acetyl phosphate transferase and acetate kinase. In an E. coil host cell, genes that are downregulated, disrupted, or deleted can include adhE, ldhA, poxB, and ackA-pta.
Further, a cell engineered for the production of flavonoids or anthocyanins can have one or more genes encoding thioesterases downregulated, disrupted, or deleted to prevent hydrolysis of precursors malonyl-CoA, acetyl-CoA, and/or p-coumaryol-CoA. Acyl-CoA
thioesterase enzymes (ACOTs) catalyze the hydrolysis of acyl-CoAs (short-, medium-, long-and very long-chain), bile acid-CoAs, and methyl branched-CoAs, to the free fatty acid and coenzyme A. For example, in an E. coil host one or more of the thioesterase genes tesA, tesB, yciA, and/or ybgC
can be downregulated, disrupted, or deleted.
In further embodiments, a cell engineered for the production of flavonoids or anthocyanins can have one or more of fatty acid degradation genes downregulated, disrupted, or deleted to improve precursor supply to the flavonoid pathway. In E. coil, for example, the acyl-coenzyme A dehydrogenase (fade) gene encoding acyl-CoA dehydrogenase, adhesion A (fadA) gene encoding 3-ketoacyl-CoA thiolase, and/or gene encoding fatty acid oxidation complex subunit alpha (fadB) can be downregulated, disrupted, or deleted.

Alternatively, or in addition, genes encoding enzymes of the tricarboxylic acid cycle (TCA), such as succinate dehydrogenase, can be disrupted or downregulated to increase alpha-ketoglutarate supply which serves as a cofactor for the flavonoid and anthocyanin pathway enzymes. Other TCA enzymes that can be downregulated include citrate synthase that converts acetyl-CoA to citrate.
Also considered, in further embodiments, is an engineered host cell for the production of flavonoids or anthocyanins to upregulate the endogenous biosynthesis of amino acid tyrosine.
Tyrosine is one of the precursors for the flavonoid biosynthesis and its conversion to 4-coumaric acid is the first committed step of the pathway. Efficient biosynthesis of L-tyrosine from feedstock such as glucose or glycerol is necessary to make biological production economically viable. L-tyrosine is one of the three aromatic amino acids derived from the shikimate pathway.
The shikimate pathway is the central metabolic route leading to formation of tryptophan (TRP), tyrosine (TYR), and phenylalanine (PHE), this pathway exclusively exists in plants and microorganisms. It starts with the condensation of intermediates of glycolysis and pentosephosphate-pathway, phosphoenolpyruvate (PEP), and erythrose-4-phosphate (E4P), respectively, which enter the pathway through a series of condensation and redox reactions via 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP), 3-dehydroquinate (DHQ), 3-dehydroshikimate (DHS) to shikimate. From there the central branch point metabolite chorismate is obtained via shikimate-3-phosphate under ATP hydrolysis and introduction of a second PEP.
The initial step of the shikimate pathway is catalyzed by DAHP synthase isozymes and regulated through feedback-inhibition. In E. coil three DAHP synthase isozymes exist (aroF, aroG, aroH), which are each feedback inhibited by one of the three aromatic amino acids (TYR, PHE, TRP), in contrast the two DAHP synthases of plants are not subject to feedback-inhibition. In plants and bacteria, the subsequent five steps are catalyzed by single enzymes. From the central intermediate chorismate the pathway branches off to anthranilate and prephenate leading to aromatic amino acid, para-hydroxybenzoic acid (pHBA) and para-aminobenzoic acid (pABA) synthesis, the latter being a precursor for folate metabolism. Strategies to increase L-tyrosine production can include, without limitation, transcriptional deregulation, removing feedback inhibition, overexpression of rate-limiting enzymes, and/or deletion of the L-phenylalanine branch of the aromatic acid biosynthetic pathway. For example, in an E. coil host the tyrR gene can be disrupted, feedback-inhibition-resistant versions of the DAHP synthase (aroG) and chorismate mutase (tyrA) can be introduced, and/or rate-limiting enzymes, shikimate kinase (aroK or aroL) and quinate (QUIN)/shikimate dehydrogenase (ydiB) can be overexpressed.
Further, the ppsA, aroG, and/or transketolase (tktA) can be overexpressed or exogenously introduced to enhance tyrosine production.
Also considered, in further embodiments, is an engineered host cell for the production of flavonoids or anthocyanins further engineered to upregulate the endogenous biosynthesis of cofactor heme. Cytochrome P450 (CYPs), one of the exogenous genes in the engineered cells provided herein, contain heme as a cofactor. Improving heme supply can be an effective strategy to increase flavonoid biosynthesis. 5-aminolevulinic acid (ALA) is the first committed precursor to the heme pathway. There exist two known alternate routes by which this committed intermediate is generated. One route is the C4 pathway (Shemin pathway), which involves the condensation of succinyl-CoA and glycine to D-aminolevulinic acid by ALA
synthase (ALAS).
The C4 pathway is restricted to mammals, fungi and purple nonsulfur bacteria.
The second route is the C5 pathway, which involves three enzymatic reactions resulting in the biosynthesis of ALA from the five-carbon skeleton of glutamate. The C5 pathway is active in most bacteria, all archaea and plants. Seven additional reactions, including assembly of eight ALA molecules into a cyclic tetrapyrrole, modification of the side chains, and incorporation of reduced iron into the molecule, are required to convert ALA to heme. In an E. coil host, the three enzymes involved in ALA biosynthesis are glutamyl-tRNA synthetase (G1tX), glutamyl-tRNA reductase (hemA), and glutamate-l-semialdehyde aminotransferase (hemL). In an E. coil host, the engineered cells provided herein can be further engineered to express or overexpress hemA or its variants, and/or hemL to increase the heme precursor ALA production. The nonlimiting examples of hemA gene that can be overexpressed include, without limitation, a mutated hemA gene from Salmonella typhimurium (EC:1.1.1.70, SEQ ID NO: 21) and hemA with SEQ ID NOS: 91-93.
Alternatively, or in addition, a heterologous ALAS gene can be introduced to produce ALA via the C4 pathway. Nonlimiting examples of heterologous ALAS that can be expressed in E.
coil include ALAS of Rhodobacter capsulatus (SEQ ID:22), ALAS with SEQ ID NOS: 94-97, or an ALAS
having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of these or other naturally-occurring ALAS. Further, one or more of the downstream genes (E. coil hemB, hemC, hemD, hemE, hemF, hemG, hemI, or hemH) that catalyze the synthesis of heme from ALA can be overexpressed to drive the flux from ALA to heme production. Cultures of cells engineered for the production of flavonoids or anthocyanins can in some embodiments include a medium that includes succinate and/or glycine, precursors of heme biosynthesis via the C4 pathway.
Engineered cells that produce a flavonoid can be engineered to include multiple pathways to enhance flavonoid production. Those skilled in the art will recognize that the embodiments described herein can be combined in multiple ways. Examples of engineered cells having multiple genetic modifications are exemplary only and do not limit the scope of the invention.
Enzymes to be expressed or overexpressed in engineered cells according to the invention are set forth in Table 11.
HOST CELLS
A host cell as provided herein can be a prokaryotic cell or a eukaryotic cell.
Eukaryotic cells may be microbial eukaryotic cells, such as, for example, fungal cells or yeast cells.
Prokaryotic cells that can be engineered as provided herein include bacterial cells and cyanobacterial cells.
Host can be selected based on their ability to take up and utilize particular carbon sources, nitrogen sources, or precursor molecules or may be engineered to take up and utilize molecules that may be added to the culture medium.
Nonlimiting examples of suitable microbial hosts for the bio-production of a flavonoid include, but are not limited to, any gram-negative organisms, more particularly a member of the family Enterobacteriaceae, such as E. coil, any gram-positive microorganism, for example Bacillus subtilis, Lactobacillus sp. or Lactococcus sp.; a yeast, for example Saccharomyces cerevisiae, Pichia pastoris or Pichia stipitis; and other groups or microbial species. More particularly, suitable microbial hosts for the bio-production of a flavonoid generally include, but are not limited to, members of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula, and Saccharomyces.

CULTURE MEDIUM
In yet another aspect, methods for producing a flavonoid or an anthocyanin that include incubating a culture of an engineered host cell as provided herein to produce a flavonoid or an anthocyanin. The methods can further include recovering the flavonoid or anthocyanin from the culture medium, whole culture, or cells.
The culture comprises cells engineered for the production of flavonoids or anthocyanins in a culture medium. In various embodiments the engineered cells can be prokaryotic or eukaryotic cells. The culture medium includes at least one carbon source that is also an energy source. Exemplary carbon sources include glucose, glycerol, sucrose, fructose, and xylose. Such carbon sources may be purified or crude, including a biomass comprising glycerol, for example, crude glycerol produced as a byproduct of biodiesel production from corn waste. In addition, the culture medium can include one or more other carbon sources or compounds to increase precursor generation or cofactor supply such as, without limitation, tyrosine, phenylalanine, coumaric acid, acetate, malonate, succinate, glycine, bicarbonate, biotin, naringenin, 5-aminolevulinic acid, thiamine, pantothenate, alpha-ketoglutarate, and ascorbate. In some embodiments, tyrosine and coumaric acid are provided in the culture medium. In some embodiments, tyrosine, alpha-ketoglutarate, 5-aminolevulinic acid, and ascorbate are provided in the culture medium.
Culture conditions can include aerobic, microaerobic or any combination alternating aerobic/microaerobic growth conditions. Further, culture conditions can include shake flasks, fermentation, and other large scale culture procedures. An exemplary growth condition for achieving a flavonoid product include aerobic or microaerobic fermentation conditions. The culture conditions can be scaled up and grown continuously for manufacturing flavonoid product. Exemplary growth procedures include, for example, fed-batch fermentation and batch separation. In an exemplary batch fermentation protocol, the cells are grown in a bioreactor that is well controlled for growth temperature, oxygen, pH, carbon sources, and other compounds.
The desired temperature can be from, for example, 20-37 C, depending on the growth characteristics of the production cells and desired conditions for the fermented products. The pH
of the bioreactor can be controlled to range from 5-8 or left uncontrolled in some cases. The batch fermentation period can last in the range of several hours to several days, for examples, 8 to 96 hours. Upon completion of the cultivation period, the fermenter contents can be passed through a cell separation unit to remove cells and cell debris. The cells can be lysed or disrupted enzymatically or chemically prior to or after separation of cells from the fermentation broth, as desired, in order to release additional product. To purify the flavonoids and/or anthocyanins to homogeneity the solution containing the flavonoids and/or anthocyanins was concentrated and the product purified via ion exchange or silica-based chromatography. The resulting solution was either lyophilized to yield the products in a solid form or was concentrated into a liquid solution.
In some embodiments, a method of producing a flavonoid or an anthocyanin comprises culturing an engineered cell disclosed herein in a culture medium to produce a flavonoid or an anthocyanin. In some embodiments, glycerol is used as a carbon feedstock. In some embodiments, the glycerol is crude glycerol. In some embodiments, the method comprises isolating naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside. In some embodiments the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, or greater than 95%. In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 50% to about 99%, e.g., from about 50% to about 95% (for example from: about 50%, 55%, 60%, 65%, 70%, 75%, 80% to about: 85%, 90%, 95%, 97.5%, 99% or 99.9%). In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 50% to: about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 55% to: about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 60% to: about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 65% to: about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 70% to: about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 75% to: about 80%, about 85%, about 90%, about 95%, or about 99%, from about 80% to about 85%, about 90%, about 95%, or about 99%.
In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 85% to: about 90%, about 95%, or about 99%. In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 90% to about 95%, or about 99%, or from about 95% to about 99% or greater.
VIII. EXAMPLES
USING THE MODIFIED CELL TO CREATE PRODUCTS
Example 1 - Production of naringenin in E. coli An E. coil cell derived from MG1655 was engineered to overexpress ACC (SEQ ID
NO:
15), TAL (SEQ ID NO: 1), 4CL (SEQ ID NO: 4), CHS (SEQ ID NO: 5), and CHI (SEQ
ID NO:
6) to produce naringenin when substrates tyrosine and coumaric acid were supplied in culture medium. ACC was expressed on a medium-copy plasmid (15-20 copies) while TAL, 4CL, CHS, and CHI were expressed on the chromosome. Cells of an OD 2.5 were cultured in a 48-well plate at 30 degree for 24 hours with a shaking speed of 600 RPM in minimal medium supplied with trace element, vitamins, 1 mM tyrosine,1 mM coumaric acid, and 2% glycerol.
Cell cultures were extracted with DMSO at 1:1 ratio and centrifuged for 15 mins. The supernatant was analyzed for naringenin with HPLC. The cells produced 232 i.tM naringenin.
Variants of the foregoing host cell may be prepared using one or more of ACC
(SEQ ID NO:
15), TAL (SEQ ID NO: 1), 4CL (SEQ ID NO: 4), CHS (SEQ ID NO: 5), and CHI (SEQ
ID NO:
6) with one or more homologs of ACC (SEQ ID NO: 15), TAL (SEQ ID NO: 1), 4CL
(SEQ ID
NO: 4), CHS (SEQ ID NO: 5), or CHI (SEQ ID NO: 6), or combinations of two or more thereof, wherein the homologous enzymes have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the referenced enzymes.
Example 2 - Production of dihydrokaempferol in E. coli An E. coil cell derived from MG1655 was engineered to overexpress F3H (SEQ ID
NO:
7) on the chromosome to produce dihydrokaempferol when substrate naringenin was supplied in culture medium. Cells of an OD 0.5-0.7 were cultured in a 24-well plate at 30 degree for 18 hours with a shaking speed of 200 RPM in minimal medium supplied with 2%
glycerol, trace elements, 0.8 mM naringenin, 65 mg/L 5-aminoleuvinic acid, 0.1 mM ferrous sulfate, 0.1 mM 2-oxoglutarate, and 2.5 mM ascorbic acid. Cell cultures were extracted with DMSO
and centrifuged for 15 minutes. The supernatant was analyzed for dihydrokaempferol with HPLC.
The cells produced 315 i.tM dihydrokaempferol.
Variants of the foregoing host cell may be prepared using a homolog of F3H
(SEQ ID NO:
7), wherein the homologous enzyme has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the referenced enzyme.

Example 3 - Production of taxifolin in E. coli An E. coil strain derived from MG1655 was engineered to overexpress F3H (SEQ
ID
NO: 7), F3'H (SEQ ID NO: 8), and CPR (SEQ ID NO: 9) to produce taxifolin when the substrate naringenin was supplied in culture medium. F3H was overexpressed on the chromosome while F3'H and CPR were overexpressed on a medium-copy plasmid. Cells of an OD 0.5-0.7 were cultured in a 24-well plate at 30 degree for 18 hours with a shaking speed of 200 RPM in minimal medium supplied with 2% glucose, 0.8 mM naringenin, 65 mg/L 5-aminoleuvinic acid, 0.1 mM ferrous sulfate, 0.1 mM 2-oxoglutarate, and 2.5 mM ascorbic acid. Cell cultures were extracted with 50% DMSO and centrifuged for 15 minutes. The supernatant was analyzed for taxifolin with HPLC. The cells produced 500 [tM taxifolin.
Variants of the foregoing host cell may be prepared using one or more of F3H
(SEQ ID NO:
7), F3'H (SEQ ID NO: 8), and CPR (SEQ ID NO: 9) along with one or more homologs of F3H
(SEQ ID NO: 7), F3'H (SEQ ID NO: 8), and CPR (SEQ ID NO: 9), or combinations of two or more thereof, wherein the homologous enzymes have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the referenced enzymes.
Example 4 - Production of anthocyanidins and anthocyanins An E. coil strain derived from MG1655 was engineered to overexpress ANS (SEQ
ID
NO: 13) and 3GT (SEQ ID NO: 14) to produce cyanidin-3-0-glucoside when the substrate (+)-catechin was supplied in culture medium. ANS and 3GT were overexpressed on the chromosome. Cells of an OD 0.5-0.7 were cultured in a 24-well plate at 30 degree for 18 hours with a shaking speed of 200 RPM in minimal medium supplied with 1.0% glucose, 2.0 mM (+)-catechin, 0.1 mM 2-oxoglutarate, and 2.5 mM ascorbic acid. Cell cultures were acidified with 2M HCL and extracted with 100% Ethanol. The supernatant was analyzed for cyanidin-3-0-glucoside by HPLC. The cells produced 50 mg/L cyanidin-3-0-glucoside.
Variants of the foregoing host cell may be prepared using one or both of ANS
(SEQ ID
NO: 13) and 3GT (SEQ ID NO: 14) along with a homolog of ANS (SEQ ID NO: 13), 3GT (SEQ
ID NO: 14), or both, wherein the homologous enzymes have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the referenced enzymes.
ANALYTICAL METHODS
Example 5 - Flavonoid precursors and flavonoids For sampling naringenin, eriodictyol, dihydrokaempferol and taxifolin, extraction of total flavonoids from E. coil were performed on whole cell broth. 500 tL of whole cell broth was vortexed for 30 seconds with 500 tL of DMSO (dimethyl sulfoxide) and centrifuged for 15 minutes. For HPLC analysis, 50 tL of supernatant was transferred to an HPLC
vial.
The HPLC method was as follows: An Agilent 1200 HPLC was fitted with an Ascentis C18 Column 150 mm X 4.6 mm, 3 p.m, equipped with a R-18 (3 p.m) guard column.
The column was heated to 30 C with the sample block being maintained at 25 C. For each sample, 5 was injected and the product was eluted at a flow rate of 1.5 mL/min using 0.1% phosphoric acid in water (solvent A), acetonitrile (solvent B), and methanol (solvent C) with the following gradient:
Tim A(%) B(%) (%) 2.5 85 10 5 7.5 70 25 5 12.5 50 45 5 The run time was a total of 15 minutes with naringenin, eriodictyol, dihydrokaempferol and taxifolin eluting at 12.50, 11.56, 10.20, and 8.85 minutes respectively. A
diode array detector (DAD) was used for the detection of the molecule of interest at 288 nm.
Example 6 - Anthocyanidins and anthocyanins For sampling (+)-catechin, cyanidin, and cyanidin-3-glucoside the reaction fluid was acidified with 13 M HC1 (1:40 v/v), and extracted with 100% ethanol followed by mixing, centrifugation and filtration through a 0.45 [tm filter. The HPLC method was as follows: An Agilent 1200 HPLC was fitted with a LiChrospher RP-8 Column 250 mm X 4.6 mm, 5 [tm, equipped with a LiChrospher 100 RP-8 (5 [tm) LiChroCART 4-4 guard column. The column was heated to 25 C with the sample block being maintained at 25 C. For each sample, 10 1..t.L
was injected and the product was eluted at a flow rate of 1.0 ml/min using 0.1% phosphoric acid in water (solvent A) and acetonitrile (solvent B) with the following gradient:
90% A to 10% A
for 12 min, 90% A for 0.5 min, and 90% A for 3.5 min for column equilibration.
The run time was a total of 16 minutes with cyanidin-3-glycoside eluting at 6.95 mins and cyanidin eluting at 8.9 minutes. A diode array detector (DAD) was used for the detection of the molecule of interest at either 280 nm or 530 nm.
Example 7¨ Flavonoid Production The example provides a combination of modifications to the E. coil host genome including deletions and overexpression of enzymes from other organisms to recapitulate the bioproduction pathway described in Figure 4. Accordingly, the invention provides an engineered host cell that comprises one or more genetic modifications (as shown in FIG. 4 and described in this Example 7 and herein above in this application) that result in production of flavonoid or anthocyanin from a carbon source that can also be an energy source, through multiple chemical intermediates, by the engineered host cell. In certain embodiments, the production of flavonoid or anthocyanin from a carbon source that can also be an energy source occurs through enzymatic transformation. In certain embodiments, the carbon source is selected from a group consisting of:
(i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, and (v) any combination thereof. In certain embodiments, the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, and (v) any combination thereof. As shown in FIG. 4, in certain embodiments, one or more genetic modifications lead to increase in metabolic flux to flavonoid precursors or cofactors. As shown in FIG. 4, in certain embodiments, one or more of the genetic modifications cause reduction of formation of byproducts. As shown in FIG. 4, in certain embodiments, one or more genetic modifications are selected from: (i) one or more modifications for over-expressing one or more endogenous genes in the engineered host cells; (ii) one or more modifications for under-expressing one or more endogenous genes in the engineered host cells;
(iii) one or more genetic modification is expressing one or more non-native genes in the engineered host cells; and (iv) a combination thereof.
As shown in FIG. 4, in certain embodiments, the engineered host cell is cultured in a medium comprising molecules selected from: tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid.
As shown in FIG. 4, in certain embodiments, the engineered host cell comprises at least one or more nucleic acid sequences selected from: (i) a nucleic acid sequences encoding tyrosine ammonia lyase activity; (ii) a nucleic acid sequences encoding phenylalanine ammonia lyase activity; (iii) cinnamate 4-hydroxylase; and (iv) any combination thereof As shown in FIG. 4, in certain embodiments, the engineered host cell comprises at least one or more peptides selected from: (i) chalcone isomerase; (ii) chalcone synthase; (iii) a fusion protein comprises a chalcone synthase and a chalcone isomerase; and (iv) any combination thereof.
As shown in FIG. 4, in certain embodiments, one or more genetic modifications decreases fatty acid biosynthesis. As shown in FIG. 4, in certain embodiments, the engineered host cell comprises an exogenous nucleic acid sequence selected from: (i) nucleic acid sequence encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid;
(iv) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (v) any combinations thereof As shown in FIG. 4, in certain embodiments, the engineered host cell comprises at least one or more nucleic acid sequences selected from: (i) nucleic acid sequences encoding tyrosine ammonia lyase activity; (ii) nucleic acid sequences encoding phenylalanine ammonia lyase activity; (iii) nucleic acid sequences encoding cinnamate 4-hydroxylase activity; (iv) nucleic acid sequences encoding 4-courmarate-CoA ligase (4CL) activity; and (v) any combination thereof.

As shown in FIG. 4, in certain embodiments, the engineered host cell comprises an exogenous nucleic acid sequence selected from the group consisting of: (i) nucleic acid sequence encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine .. ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid;
(iv) nucleic acid sequence encoding 4-courmarate-CoA ligase activity, wherein 4-courmarate-CoA
ligase forms p-coumaroyl-CoA from coumaric acid (v) nucleic acid sequence encoding chalcone synthase .. activity, wherein chalcone synthase forms naringenin chalcone from malonyl-CoA and p-coumaroyl-CoA; (vi) nucleic acid sequence encoding chalcone isomerase activity, wherein chalcone isomerase forms naringenin from naringenin chalcone; (vii) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (viii) any combinations thereof The compositions as described above, can be used in methods described herein for increasing the production of flavonoids or anthocyanins. Such methods involve providing any of the compositions described above to result in enzymatic transformation by the engineered host cell of glycerol through multiple chemical intermediates into a flavonoid or anthocyanin (such as shown in part or in whole in FIG. 4).
In yet another aspect, it is envisioned that the pathway illustrated in FIG. 4 can be carried out using a plurality of engineered host cells, as opposed to a single host cell as described above.
In such embodiments, the plurality of the engineered host cells have one or more genetic modifications that result in enzymatic transformation by the engineered host cell of glycerol through multiple chemical intermediates into a flavonoid or anthocyanin (as shown in FIG. 4).
Aspects of the invention are now described with reference herein to FIG. 4.
Step 1: conversion of pyruvate to acetate. poxB is deleted to reduce carbon loss and eliminate the byproducts.
Step 2: conversion of pyruvate to lactate. ldhA is deleted to reduce carbon loss and eliminate the byproducts.

Step 3: conversion of Acetyl-CoA to acetate. ackA-pta is deleted to reduce carbon loss and eliminate the byproducts.
Step 4: conversion of Acetyl-CoA to ethanol (Et0H). adhE is deleted to reduce carbon loss and eliminate the byproducts.
Step 5: conversion of acetyl-CoA to a substrate for the tricarboxylic acid cycle (TCA).
Step 6: conversion of acetyl-CoA to mal-CoA. Heterologous ACC is expressed to increase the concentration of available mal-CoA. Heterologous ACC may be obtained from fungal species. Accordingly, embodiments of the invention provide an engineered host cell that comprises one or more genetic modifications to increase the production and/or availability of malonyl-CoA. In certain embodiments, the engineered host cell comprises one or more genetic modifications selected from: (i) expression of acetyl-CoA carboxylase (ACC);
and (ii) overexpression of acetyl-CoA carboxylase. In another embodiment, the engineered host cell is an E. coil. In certain embodiments, the acetyl-CoA carboxylase is from: Mucor circinelloides, Rhodotorula toruloides, Lipomyces starkeyi, and Ustilago maydis, and orthologs of acetyl-CoA
carboxylase having at least 50% amino acid identity to the acetyl-CoA
carboxylase of these aforementioned species. In certain embodiments, one or more genetic modification is deletion or attenuation of one or more fatty biosynthetic genes resulting in decrease in fatty acid biosynthesis. In certain embodiments, one or more genetic modification is overexpression of acetyl-CoA synthase (ACS). In certain embodiments, the acetyl-CoA synthase is selected from:
acetyl-CoA synthase gene of E. coil, acetyl-CoA synthase gene of Salmonella Ophimurium, and orthologs of acetyl-CoA synthase gene in any other species having at least 50%
amino acid identity to the acetyl-CoA synthase gene of E. coil and Salmonella typhimurium. In certain embodiments, one or more genetic modification is selected from a group consisting of: (i) overexpression a gene encoding pyruvate dehydrogenase (PDH), wherein the PDH
may include E354K mutation; (ii) exogenous nucleic acid sequence encoding a malonyl-CoA
synthetase; (iii) upregulation of endogenous pantothenate kinase (PanK), wherein PanK is not feedback inhibited by coenzyme A; (iv) exogenous nucleic acid sequence encoding a malonate transporter; and (v) any combinations thereof In certain embodiments, the malonyl-CoA synthetase is selected from of malonyl-CoA synthetases of Streptomyces coelicolor, Rhodopseudomonas palustris, or a malonyl-CoA synthetase having at least 50% identity to any of these or other naturally occurring malonyl-CoA synthetases. In certain embodiments, one or more genetic modifications to decrease fatty acid biosynthesis is selected from: (i) mutation or downregulation of a gene encoding malonyl-CoA-ACP transacylase (E. coil fabD); (ii) modifications to the gene beta-ketoacyl-ACP synthase II (E. coil fabF); (iii) downregulation of beta-ketoacyl-ACP synthase I
enzyme (E. coil fabB); (iv) downregulation of acyl carrier protein (E. coil acpP); and (v) any combinations thereof. In certain embodiments, the engineered host cell comprises peptides selected from: (i) acetyl-CoA carboxylase (ACC) having an amino acid sequence at least 80%
identical to the polypeptide set forth in SEQ ID NO: 15 or SEQ ID NO: 16; (ii) malonate CoA-transferase having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 19; (iii) acetyl-CoA synthase (ACS) having an amino acid sequence at least 80%
identical to the polypeptide set forth in SEQ ID NO: 16; (iv) malonyl-CoA
synthase having an amino acid sequence at least 80% identical SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO:
79; (v) malonate transporter having an amino acid sequence at least 80%
identical to SEQ ID
NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO:
85, SEQ ID NO: 86, or SEQ ID NO: 87; (vi) pantothenate kinase having an amino acid sequence at least 80% identical to SEQ ID NO: 88, SEQ ID NO: 89, or SEQ ID NO: 90; and (vii) any combinations thereof.
In another aspect, the invention provides a method of increasing the production of flavonoids comprising an engineered host cell, wherein the one or more engineered host cells comprise one or more genetic modifications to increase production and/or availability of malonyl-CoA. In certain embodiments, the engineered host cell comprises one or more genetic modifications selected from: (i) expression of acetyl-CoA carboxylase (ACC);
and (ii) overexpression of acetyl-CoA carboxylase. In another embodiment, the engineered host cell is an E. coil. In certain embodiments, the acetyl-CoA carboxylase is from: Mucor circinelloides, Rhodotorula toruloides, Lipomyces starkeyi, and Ustilago maydis, and orthologs of acetyl-CoA
carboxylase having at least 50% amino acid identity to the acetyl-CoA
carboxylase of these aforementioned species. In certain embodiments, one or more genetic modification is deletion or attenuation of one or more fatty biosynthetic genes resulting in decrease in fatty acid biosynthesis. In certain embodiments, one or more genetic modification is overexpression of acetyl-CoA synthase (ACS). In certain embodiments, the acetyl-CoA synthase is selected from:
acetyl-CoA synthase gene of E. coil, acetyl-CoA synthase gene of Salmonella Ophimurium, and orthologs of acetyl-CoA synthase gene in any other species having at least 50%
amino acid identity to the acetyl-CoA synthase gene of E. coil and Salmonella typhimurium. In certain embodiments, one or more genetic modification is selected from a group consisting of: (i) overexpression a gene encoding pyruvate dehydrogenase (PDH), wherein the PDH
may include E354K mutation; (ii) exogenous nucleic acid sequence encoding a malonyl-CoA
synthetase; (iii) upregulation of endogenous pantothenate kinase (PanK), wherein PanK is not feedback inhibited by coenzyme A; (iv) exogenous nucleic acid sequence encoding a malonate transporter; and (v) any combinations thereof In certain embodiments, the malonyl-CoA synthetase is selected from of malonyl-CoA synthetases of Streptomyces cod/color, Rhodopseudomonas palustris, or a malonyl-CoA synthetase having at least 50% identity to any of these or other naturally occurring malonyl-CoA synthetases. In certain embodiments, one or more genetic modifications to decrease fatty acid biosynthesis is selected from: (i) mutation or downregulation of a gene encoding malonyl-CoA-ACP transacylase (E. coil fabD); (ii) modifications to the gene beta-ketoacyl-ACP synthase II E. coil fabF); (iii) downregulation of beta-ketoacyl-ACP synthase I
enzyme (E. coil fabB); (iv) downregulation of acyl carrier protein (E. coil acpP); and (v) any combinations thereof. In certain embodiments, the engineered host cell comprises peptides selected from: (i) acetyl-CoA carboxylase (ACC) having an amino acid sequence at least 80%
identical to the polypeptide set forth in SEQ ID NO: 15 or SEQ ID NO: 16; (ii) malonate CoA-transferase having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 19; (iii) acetyl-CoA synthase (AC S) having an amino acid sequence at least 80%
identical to the polypeptide set forth in SEQ ID NO: 16; (iv) malonyl-CoA
synthase having an amino acid sequence at least 80% identical SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO:
79; (v) malonate transporter having an amino acid sequence at least 80%
identical to SEQ ID
NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO:
85, SEQ ID NO: 86, or SEQ ID NO: 87; (vi) pantothenate kinase having an amino acid sequence at least 80% identical to SEQ ID NO: 88, SEQ ID NO: 89, or SEQ ID NO: 90; and (vii) any combinations thereof.
Step 7: conversion of mal-CoA to malonyl-ACP (acyl carrier protein). malonyl-coA-ACP
transacylase (fabD) is downregulated to increase carbon flux.

Step 8: conversion of malonyl-ACP to 3-ketyoacyl-ACP. beta-ketoacyl-ACP
synthase II
(fabF) is downregulated to increase carbon flux.
Step 9: conversion to mal-CoA to naringenin chalcone; conversion of coumaryl-CoA to naringenin chalcone. A heterologous CHS is overexpressed.
Step 10: conversion to naringenin chalcone to naringenin. A heterologous CHI
is overexpressed.
Steps 11, 12, and 13: conversion of naringenin to dihydrokaempferol (DHK);
conversion of naringenin to eriodictyol (EDL); conversion of eriodictyol (EDL) to dihydroquercetin (DHQ);
conversion of (DHK) to dihydroquercetin (DHQ); conversion of dihydrokaempferol (DHK) to dihydromyricetin (DHM); conversion of pentahydroxyflayaone (PHF) to dihydromyricein (DHM). Heterologous F3'5'H, F3H, F3H, and/or CPR are overexpressed.
Accordingly, as shown in FIG. 4, in another aspect, the invention provides method of increasing the production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictyol (EDL), and/or pentahydroxyflayaone (PHF) comprising an engineered host cell, wherein the engineered host cell comprises cytochrome P450 reductase (CPR) and at least one of flavanone-3'-hydroxylase (F3'H) or flavonoid 3',5'-hydroxylase (F3'5'H). In certain embodiments, the precursor for increase in production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictyol (EDL), and/or pentahydroxyflavone (PHF) is naringenin and/or dihydrokaempferol (DHK).
In certain embodiments, the engineered host cell further comprises peptides selected from a group consisting of: (i) flavonoid 3'-hydroxylase (F3'H); (ii) cytochrome P450 reductase (CPR); and (iii) any combination thereof In certain embodiments, the engineered host cell produces eriodictyol or taxifolin. In certain embodiments, the engineered host cell further comprises flavonoid 3',5'-hydroxylase (F3'5'H). In certain embodiments, the engineered host cell produces pentahydroxyflavone or dihydromyricetin. In certain embodiments, flavonoid 3'-hydroxylase (F3'H) is truncated to remove the N-terminal leader sequence. In certain embodiments, cytochrome P450 reductase (CPR) is truncated to remove the N-terminal leader sequence. In certain embodiments, flavonoid 3'-hydroxylase (F3'H) is fused with cytochrome P450 reductase (CPR). In certain embodiments, flavonoid 3',5'-hydroxylase (F3'5'H) is fused with cytochrome P450 reductase (CPR). In certain embodiments, flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID
NO. 7. In certain embodiments, flavanone-3'-hydroxylase (F3'H) has an amino acid sequence at least 80%
identical to the polypeptide set forth in SEQ ID NO. 8. In certain embodiments, cytochrome P450 reductase (CPR) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 9. In certain embodiments, flavonoid 3',5'-hydroxylase (F3'5'H) has an amino acid sequence at least 80% identical to the polypeptides selected from a group consisting of: (i) SEQ ID NO. 10, (ii) SEQ ID NO. 56, and (iii) SEQ ID NO. 57. In certain embodiments, the engineered host cell further comprises cytochrome b5. In certain embodiments, cytochrome b5 has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO.
98.
As shown in FIG. 4, in another aspect, the invention provides method of increasing the production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictyol (EDL), and/or pentahydroxyflayaone (PHF) comprising an engineered host cell, wherein the engineered host cell comprises cytochrome P450 reductase (CPR) and at least one of flavanone-3'-hydroxylase (F3'H) or flavonoid 3',5'-hydroxylase (F3'5'H). In certain embodiments, the precursor for increase in production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictyol (EDL), and/or pentahydroxyflavone (PHF) is naringenin and/or dihydrokaempferol (DHK).
In certain embodiments, the engineered host cell further comprises peptides selected from a group consisting of: (i) flavonoid 3'-hydroxylase (F3'H); (ii) cytochrome P450 reductase (CPR); and (iii) any combination thereof In certain embodiments, the engineered host cell produces eriodictyol or taxifolin. In certain embodiments, the engineered host cell further comprises flavonoid 3',5'-hydroxylase (F3'5'H). In certain embodiments, the engineered host cell produces pentahydroxyflavone or dihydromyricetin. In certain embodiments, flavonoid 3'-hydroxylase (F3'H) is truncated to remove the N-terminal leader sequence. In certain embodiments, cytochrome P450 reductase (CPR) is truncated to remove the N-terminal leader sequence. In certain embodiments, flavonoid 3'-hydroxylase (F3'H) is fused with cytochrome P450 reductase (CPR). In certain embodiments, flavonoid 3',5'-hydroxylase (F3'5'H) is fused with cytochrome P450 reductase (CPR). In certain embodiments, flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID
NO. 7. In certain embodiments, flavanone-3'-hydroxylase (F3'H) has an amino acid sequence at least 80%
identical to the polypeptide set forth in SEQ ID NO. 8. In certain embodiments, cytochrome P450 reductase (CPR) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 9. In certain embodiments, flavonoid 3',5'-hydroxylase (F3'5'H) has an amino acid sequence at least 80% identical to the polypeptides selected from a group consisting of: (i) SEQ ID NO. 10, (ii) SEQ ID NO. 56, and (iii) SEQ ID NO. 57. In certain embodiments, the engineered host cell further comprises cytochrome 13,5. In certain embodiments, cytochrome b5 has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO.
98.
Step 14: conversion of dihydroquercetin (DHQ) to leucocyanidin (LC);
conversion of dihydrokaempferol (DHK) to leucopelargonidin (LP); and conversion of dihydromyricetin (DHM) to leucodelphinidin (LD). Heterologous DFR is overexpressed.
Step 15: conversion of leucocyanidin (LC) to catechin; conversion of leucodelphinidin (LD) to gallocatechin; and conversion of leucopelargonidin (LP) to afzelechin.
Heterologous LAR is overexpressed.
Step 16: conversion of catechin to cyanidin; conversion of leucocyanidin (LC) to catechin; conversion to leucodelphinidin (LD) to delphinidin; conversion of gallocatechin to delphinidin; conversion of leucopelargonidin (LP) to pelargonidin; or conversion of afzelechin to pelargonidin. Heterologous ANS is overexpressed. Step 16 could be carried in vivo or in a cell-free medium. Accordingly, as shown in FIG. 4, in another aspect, the invention provides an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase transformation of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G). In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase. In certain embodiments, the anthocyanin synthase is selected from: (i) anthocyanin synthase of Car/ca papaya (SEQ. ID NO:13); (ii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 66, SEQ. ID NO: 67, SEQ. ID NO:
68, or SEQ. ID NO: 69; (iii) the anthocyanin synthase has an amino acid sequence at least 80%
identical to SEQ. ID NO: 13; and (iv) any combinations thereof In certain embodiments, one or more engineered host cells comprises flavonoid-3-glucosyl transferase (3GT).
In certain embodiments, flavonoid-3-glucosyl transferase is selected from: (i) flavonoid-3-glucosyl transferase in Vitis labrusca (SEQ. ID NO:14); (ii) the flavonoid-3-glucosyl transferase has an amino acid sequence at least 80% identical to SEQ. ID NO: 70, SEQ. ID NO: 71, SEQ. ID NO:
72, or SEQ. ID NO: 73; and (iii) any combinations thereof. In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT).
In certain embodiments, the one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, the one or more genetic modifications are selected from a group consisting of: (i) anthocyanin synthase, (ii) flavonoid-3-glucosyl transferase (3GT), and (iii) a combination thereof.
In another aspect, the invention provides a method for increasing the production of flavonoids comprising an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase transformation of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G). In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase. In certain embodiments, the anthocyanin synthase is selected from: (i) anthocyanin synthase of Car/ca papaya (SEQ. ID NO:13); (ii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 66, SEQ. ID NO: 67, SEQ. ID NO: 68, or SEQ. ID NO: 69; (iii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 13; and (iv) any combinations thereof.
In certain embodiments, one or more engineered host cells comprises flavonoid-3-glucosyl transferase (3GT). In certain embodiments, flavonoid-3-glucosyl transferase is selected from: (i) flavonoid-3-glucosyl transferase in Vitis labrusca (SEQ. ID NO:14); (ii) the flavonoid-3-glucosyl transferase has an amino acid sequence at least 80% identical to SEQ. ID NO:
70, SEQ. ID NO:
71, SEQ. ID NO: 72, or SEQ. ID NO: 73; and (iii) any combinations thereof. In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, the one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT).
In certain embodiments, the one or more genetic modifications are selected from a group consisting of: (i) anthocyanin synthase, (ii) flavonoid-3-glucosyl transferase (3GT), and (iii) a combination thereof In another aspect, the invention provides a method of increasing the transformation of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G) comprising anthocyanin synthase, wherein the anthocyanin synthase is selected from: (i) anthocyanin synthase of Car/ca papaya (SEQ. ID NO:13); (ii) the anthocyanin synthase has an amino acid sequence at least 80%
identical to SEQ. ID NO: 66, SEQ. ID NO: 67, SEQ. ID NO: 68, or SEQ. ID NO:
69; (iii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ.
ID NO: 13; and (iv) any combinations thereof.
In another aspect, the invention provides a method of increasing the transformation of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G) comprising flavonoid-3-glucosyl transferase (3GT), wherein the flavonoid-3-glucosyl transferase is selected from: (i) flavonoid-3-glucosyl transferase in Vitis labrusca (SEQ. ID NO:14); (ii) the flavonoid-3-glucosyl transferase has an amino acid sequence at least 80% identical to SEQ. ID NO: 70, SEQ. ID
NO: 71, SEQ. ID
NO: 72, or SEQ. ID NO: 73; and (iii) any combinations thereof.
Step 17: conversion of pelargonidin to callistephin; conversion of delphinidin to myrtillin (De3G); conversion of cyanidin to Cy3G. Heterologous 3GT was overexpressed in E. colt. Step 17 could be carried in vivo or as a cell-free reaction.
Step 18: conversion of pyruvate to phosphoenolpyruvate (PEP). ppsA is overexpressed to upregulate tyrosine.
Step 19: conversion of fructose-6-phosphate (F6P) to erythrose-4-phosphate (E4P). tktA
is overexpressed to upregulate tyrosine.
Step 20: conversion of phosphoenolpyruvate (PEP) to deoxy-d-arabino-heptulosonate-7-phosphate (DAHP). aroG variant is overexpressed to upregulate tyrosine.
Step 21: conversion of deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) to dehydroquinate (DHQ); conversion of erythrose-4-phosphate (E4P) to dehydroquinate (DHQ).
Step 22: conversion of dehydroquinate (DHQ) to 3-dehydroshikimate (DHS).
Step 23: conversion of 3-dehydroshikimate (DHS) to shikimic acid (SHK). aroE
is overexpressed to upregulate tyrosine.
Step 24: conversion of shikimic acid (SHK) to shikimate-3-phosphate (53P).

Step 25: conversion of shikimate-3-phosphate (53P) to 5-enolpyruvylshikimate-3-phosphate (EPSP).
Step 26: conversion of 5-enolpyruvylshikimate-3-phosphate (EPSP) to chorismic acid (CHA).
Step 27: conversion of chorismic acid (CHA) to prephenate (PPA); conversion of prephenate (PPA) to 4-hydroxy-phenylpyruvate (HPP). tryA variant is overexpressed.
Step 28: conversion of 4-hydroxy-phenylpyruvate (HPP) to tyrosine; conversion of phenylpyruvate (POPP) to phenylalanine (Phe). Accordingly, as shown in FIG. 4, embodiments of the invention provide an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase endogenous biosynthesis of tyrosine.
In certain embodiments, one or more genetic modifications comprises upregulation of 3-deoxy-D-arabino-heptulosonate synthase. In certain embodiments, one or more genetic modifications are selected from: (i) upregulation of chorismate mutase; (ii) upregulation of prephenate dehydrogenase; (iii) overexpression of shikimate kinase; (iv) overexpression of shikimate dehydrogenase; and (v) any combinations thereof In certain embodiments, one or more genetic modifications comprises downregulation of L-phenylalanine biosynthetic pathway. In certain embodiments, one or more genetic modifications comprises expression of exogenous phosphoenolpyruvate synthase (ppsA).
In certain embodiments, one or more genetic modifications comprises expression of exogenous transketolase (tktA). In certain embodiments, wherein the one or more genetic modifications comprises disruption of tyrR gene.
As shown in FIG. 4, in another aspect, the invention provides a method of increasing endogenous biosynthesis of tyrosine comprising an engineered cell, wherein the engineered host cell comprises one or more genetic modifications to increase endogenous biosynthesis of tyrosine. In certain embodiments, one or more genetic modifications comprises upregulation of 3-deoxy-D-arabino-heptulosonate synthase. In certain embodiments, one or more genetic modifications are selected from: (i) upregulation of chorismate mutase; (ii) upregulation of prephenate dehydrogenase; (iii) overexpression of shikimate kinase; (iv) overexpression of shikimate dehydrogenase; and (v) any combinations thereof. In certain embodiments, one or more genetic modifications comprises downregulation of L-phenylalanine biosynthetic pathway.
In certain embodiments, one or more genetic modifications comprises expression of exogenous phosphoenolpyruvate synthase (ppsA). In certain embodiments, one or more genetic modifications comprises expression of exogenous transketolase (tktA). In certain embodiments, wherein the one or more genetic modifications comprises disruption of tyrR
gene.
Step 29: conversion of tyrosine to coumaric acid. A heterologous TAL is overexpressed.
Step 30: conversion of courmaric acid to coumaryl-CoA. A heterologous 4CL is overexpressed.
Step 31: conversion of glutamate (Glut) to glutamyl-tRNA.
Step 32: conversion of glutamyl-tRNA to glutamate semialdehyde (GSA). hemA is overexpressed to upregulate ALA.
Step 33: conversion of glutamate semialdehyde (GSA) to 6 amino levulinic acid (ALA).
hemL is overexpressed to upregulate ALA.
Step 34: conversion of 6 amino levulinic acid (ALA) to porphobilinogen (PBG).
Step 35: conversion of porphobilinogen (PBG) to hydroxymethylbilane (HMB).
Step 36: conversion of hydroxymethylbilane (HMB) to uroporphyrinogen III
(UPPIII).
Step 37: conversion of uroporphyrinogen III (UPPIII) to coproporphyrinogen III
(CPPIII).
Step 38: conversion of coproporphyrinogen III (CPPIII) to protoporphyrinogen IX
(PPPIX).
Step 39: conversion of protoporphyrinogen IX (PPPIX) to protoporphyrin IX, which is subsequently covered to heme.
Step 40: conversion of prephenate (PPA) to phenylpyruvate (POPP).
Step 41: conversion of phenylalanine (Phe) to cinnamate. Heterologous PAL
and/or TAL
are overexpressed.
Step 42: conversion of cinnamate to coumaric acid. Heterologous C4H/CPR are overexpressed.

Table 11: Enzyme Sequences:
Enzyme: Sequence:
SEQ ID:
Tyrosine ammonia- MTQVVERQADRLSSREYLARVVRSAGWDAGLTSCTD 1 lyase (TAL) EEIVRMGA S ART IEEYLK SDKP IYGL T Q GF GPL VLF D A
D SELEQ GGSLISHL GT GQ GAPL APEV SRLILWLRIQNM
RKGYSAVSPVFWQKLADLWNKGF TP AIPRHGTV SA S
Saccharothrix GDL QPLAHAALAF T GVGEAW TRD AD GRW STVPAVD
espanaensis ALAALGAEPFDWPVREALAFVNGTGASLAVAVLNHR
SALRLVRACAVL SARLATLLGANPEHYDVGHGVARG
QVGQLTAAEWIRQ GLPRGMVRD G SRPLQEPY SLRC A
Accession:
PQVLGAVLDQLDGAGDVLAREVDGCQDNPITYEGEL
ABC88669.1 LHGGNFHAMPVGF A SD QIGLAMHMAAYLAERQL GL
LVSPVTNGDLPPMLTPRAGRGAGLAGVQISATSFVSRI
RQLVFPASLTTLPTNGWNQDHVPMALNGANSVFEAL
ELGWLTVGSLAVGVAQLAAMTGHAAEGVWAELAGI
CPPLDADRPLGAEVRAARDLL SAHADQLLVDEADGK
DFG
Phenylalanine MSQVALFEQELMLHGKHTLLLNGNDLTITDVAQMAK 2 ammonia-lyase GTFEAF TFHISEEANKRIEECNELKHEIMNQHNPIYGV
(PAL) TTGFGD S VHRQ I S GEKAWDL QRNLIRF L SCGVGPVAD
EAVARATMLIRTNCLVKGNSAVRLEVIHQLIAYMERG
ITPIIPERGSVGASGDLVPLSYLASILVGEGKVLYKGEE
Brevi bacillus REVAEAL GAEGLEPL TLEAKEGLALVNGT SFM S AF AC
laterosporus LIIIG LAYADAEEIAF IADIC TAMA SEALL GNRGHF Y SF IHEQ

SKAYLELTQ SIQDRYSIRCAPHVTGVLYDTLDWVKK
WLEVEINSTNDNPIFDVETRDVYNGGNFYGGHVVQA
Accession:
MDSLKVAVANIADLLDRQLQLVVDEKFNKDLTPNLIP
WP 00333 7219.1 RFNNDNYEIGLHEIGFKGMQIAS S AL TAEALKM S GPV S

VFSRSTEAHNQDKVSMGTISSRDARTIVELTQHVAAIH
LIALCQALDLRDSKKMSPQTTKIYNMIRKQVPFVERD
RALDGDIEKVVQLIRSGNLKKEIHDQNVND
Cinnamate-4- MDLLLIEKTLLALFAAIIGAIVISKLRGKRFKLPPGPLP 3 hydroxylase (C4H) VPIFGNWLQVGDDLNHRNLTDLAKKFGEIFLLRMGQ
RNLVVVSSPDLAKEVLHTQGVEFGSRTRNVVFDIFTG
KGQDMVFTVYGEHWRKMRRIMTVPFFTNKVVQQYR
Helianthus annuus YGWEAEAAAVVEDVKKNPAAATEGVVIRRRLQLMM
L. YNNMFRIMFDRRFESEDDPLFVKLKALNGERSRLAQS
FEYNYGDFIPILRP
FLKGYLKLCKEVKEKRFQLFKDYFVDERKKLESTKSV
Accession:
DNNQLKCAIDHILDAKEKGEINEDNVLYIVENINVAAI
QJC72299.1 ETTLWSIEWGIAELVNHPEIQAKLRNELDTKLGPGVQ
VTEPDLHKLPYLQAVIKETLRLRMAIPLLVPHMNLHD
AKLGGYDIPAESKILVNAWWLANNPEQWKKPEEFRP
ERFFEEESKVEANGNDFRYLPFGVGRRSCPGIILALPIL
GITIGRLVQNFELLPPPGQSKVDTTEKGGQFSLHILKHS
TIVAKPRAL
4-coumarate-CoA MGDCVAPKEDLIFRSKLPDIYIPKHLPLHTYCFENISKV 4 ligase (4CL) GDKSCLINGATGETFTYSQVELLSRKVASGLNKLGIQ
QGDTIMLLLPNSPEYFFAFLGASYRGAISTMANPFFTS
AEVIKQLKASQAKLIITQACYVDKVKDYAAEKNIQIIC
Petrosehnum IDDAPQDCLHFSKLMEADESEMPEVVINSDDVVALPY
crispum SSGTTGLPKGVMLTHKGLVTSVAQQVDGDNPNLYM
HSEDVMICILPLFHIYSLNAVLCCGLRAGVTILIIVIQKF
DIVPFLELIQKYKVTIGPFVPPIVLAIAKSPVVDKYDLS
Accession:
SVRTVMSGAAPLGKELEDAVRAKFPNAKLGQGYGM
P14912.1 TEAGPVLAMCLAFAKEPYEIKSGACGTVVRNAEMKIV
DPETNASLPRNQRGEICIRGDQIMKGYLNDPESTRTTI
DEEGWLHTGDIGFIDDDDELFIVDRLKEIIKYKGFQVA

PAELEALLLTHPTISDAAVVPMIDEKAGEVPVAFVVRT
NGFTTTEEEIKQFVSKQVVFYKRIFRVFFVDAIPKSPSG
KILRKDLRARIASGDLPK
Chalcone synthase MVTVEEYRKAQRAEGPATVMAIGTATPTNCVDQ STY 5 (CHS) PDYYFRITNSEHKTDLKEKFKRIVICEKSMIKKRYMHLT
EEILKENPSMCEYMAPSLDARQDIVVVEVPKLGKEAA
QKAIKEWGQPKSKITHLVFCTTSGVDMPGCDYQLTKL
Petunia x hybrida LGLRPSVKRLMMYQQGCFAGGTVLRLAKDLAENNK
GARVLVVCSEITAVTFRGPNDTHLDSLVGQALFGDGA
GAIIIGSDPIPGVERPLFELVSAAQTLLPDSHGAIDGHL
Accession:
REVGLTFHLLKDVPGLISKNIEKSLEEAFRPLSISDWNS
AAF60297.1 LFWIAHPGGPAILDQVEIKLGLKPEKLKATRNVLSNY
GNIVISSACVLFILDEMRKASAKEGLGTTGEGLEWGVL
FGFGPGLTVETVVLHSVAT
Chalcone isomerase MAASITAITVENLEYPAVVTSPVTGKSYFLGGAGERG 6 (CHI) LTIEGNFIKFTAIGVYLEDIAVASLAAKWKGKSSEELL
ETLDFYRDIISGPFEKLIRGSKIRELSGPEYSRKVMENC
Medicago sativa VAHLKSVGTYGDAEAEAMQKFAEAFKPVNFPPGASV
Accession: FYRQ SPD GIL GL SF SPDTSIPEKEAALIENKAVS SAVLE
P28012.1 TMIGEHAVSPDLKRCLAARLPALLNEGAFKIGN
Flavanone 3- MAP TP TTL TAIAGEKTL Q Q SF VRDEDERPKVAYNQF S 7 hydroxylase (F3H) NEIPIISLSGIDEVEGRRAEICNKIVEACEDWGVFQIVD
HGVDAKLISEMTRLARDFFALPPEEKLRFDMSGGKKG
GFIVS SHLQGEAVQDWREIVTYF SYPVRHRDYSRWPD
Rubus occidentalis KPEGWRAVTQQYSDELMGLACKLLEVLSEAMGLEKE
ALTKACVDMDQKVVVNFYPKCPQPDLTLGLKRHTDP
GTITLLLQDQVGGLQATRDGGKTWITVQPVEGAFVV
Accession:
NLGDHGHFLSNGRFKNADHQAVVNSNHSRLSIATFQ
ACM17897.1 NPAQEAIVYPLKVREGEKPILEEPITYTEMYKKKMSK
DLELARLKKLAKEQQPED SEKAKLEVK Q VDDIF A
Flavonoid 3' MTNLYLTILLPTFIFLIVLVLSRRRNNRLPPGPNPWPIIG 8 hy droxyl as e (F3 'H) NLPHMGPKPHQTLAAMVTTYGPILHLRLGFADVVVA
ASKSVAEQFLKVHDANFASRPPNSGAKHMAYNYQDL
VFAPYGQRWRMLRKIS SVHLF SAKALEDFKHVRQEE
Brass/ca nap 115 VGTLMRELARANTKPVNLGQLVNMCVLNALGREMI
GRRLFGADADHKAEEFRSMVTEMMALAGVFNIGDFV
PALDCLDLQGVAGKMKRLHKRFDAFLSSILEEHEAM
Accession:
KNGQDQKHTDMLSTLISLKGTDFDGEGGTLTDTEIKA
ABC58723.1 LLLNMFTAGTDTSASTVDWAIAELIRHPEIMRKAQEE
LD SVVGRGRPINESDL SQLPYLQAVIKENFRLHPPTPL S
LPHIA SE S CEINGYHIPK GS TLL TNIWAIARDPD Q W SDP
LTFRPERFLPGGEKAGVDVKGNDFELIPFGAGRRICAG
LSLGLRTIQLLTATLVHGFEWELAGGVTPEKLNIVIEET
YGITLQRAVPLVVHPKLRLDMSAYGLGSA
Cytochrome P450 MDSSSEKLSPFELMSAILKGAKLDGSNSSDSGVAVSPA 9 VMAMLLENKELVMILTTSVAVLIGCVVVLIWRRSSGS
reductase (CPR) GKKVVEPPKLIVPKSVVEPEEIDEGKKKF TIFF GT Q T GT
AEGFAKALAEEAKARYEKAVIKVIDIDDYAADDEEYE
Catharanthus EKFRKETLAFFILATYGDGEPTDNAARFYKWFVEGND
rose us RGDWLKNLQYGVFGLGNRQYEHFNKIAKVVDEKVA
EQGGKRIVPLVLGDDDQCIEDDFAAWRENVWPELDN
LLRDEDD TTVS TTYT AAIPEYRVVF PDK SD SLISEANG
Accession: HANGYANGNTVYDAQHPCRSNVAVRKELHTPASDRS

LLNLPPETYF SLHADKEDGTPLAGS SLPPPFPPCTLRTA
LTRYADLLNTPKKSALLALAAYASDPNEADRLKYLAS
PAGKDEYAQ SLVANQRSLLEVMAEFP S AKPPL GVF F A
AIAPRLQPRFYSISSSPRMAPSRIHVTCALVYEKTPGGR

IHKGVC STWMKNAIPLEESRDC SWAPIFVRQ SNFKLP
ADPKVPVIIVIIGP GT GLAPFRGFL QERLALKEEGAELGT
AVFFFGCRNRKMDYIYEDELNHFLEIGAL SELL VAF SR
EGP TKQYVQHKMAEKA SDIWRMI SD GAYVYVC GDA
KGMARDVHRTLHTIAQEQGSMD S TQAEGF VKNL QM
TGRYLRD VW
Flavonoid 3', 5'- MS T SLLLAAAAILFF ITHLFLRFLL SPRRTRKLPP GPKG 10 hydroxylase WPVVGALPMLGNMPHAALADL SRRYGPIVYLKLGSR
(F3' 5'H) GMVVASTPD SARAFLKTQDLNF SNRPTDAGATHIAYN
SQDMVFADYGPRWKLLRKL SSLHMLGGKAVEDWAV
VRRDEVGYMVKAIYES SCAGEAVHVPDMLVFAMAN
Delphinium MLGQVIL SRRVFVTKGVESNEFKEMVIELMT SA GLFN
grandiflorum VGDFIP SIAWMDLQGIVRGMKRLHKKFDALLDKILRE
HTATRRERKEKPDLVDVLMDNRDNKSEQERLTDTNI
KALLLNLF SAGTDTS S STIEWALTEMIKNP SIFGRAHA
Accession:
EMDQVIGRNRRLEESDIPKLPYLQAICKETFRKHP STP

ENPLEFNPDRFLTGKMAKIDPRGNNFELIPFGAGRRIC
AGTRMGIVLVEYILGSLVHAFEWKLRDGETLNIVIEETF
GIALQKAVPLAAVVTPRLPP SAYVV
D i hy drofl av on ol 4- M MHK GTVC VT GAAGF VGSWLIMRLLEQ GY S VKAT V 11 reductase (DFR) RDP SNMKKVKHLLDLPGAANRLTLWKADLVDEGSFD
EPIQ GC T GVFHVATPMDFE SKDPE SEMIKP TIEGMLNV
LRSCARAS STVRRVVFTS SAGTVSIHEGRRHLYDETS
Anthurium W SD VDF CRAKKMT GWMYF V SK TLAEKAAWDF AEK
andraeanum NNIDF I S IIP TL VNGPF VMP TMPP SML S AL ALITRNEPH
YSILNPVQFVHLDDLCNAHIFLFECPDAKGRYIC S SHD
VTIAGLAQILRQRYPEFDVPTEFGEMEVFDIISYSSKKL
Accession:
TDLGFEFKYSLEDMFDGAIQ SCREKGLLPPATKEP SYA
AAP20866. 1 TEQLIATGQDNGH

Leuco anthocy ani di MTV S GAIP SMTKNRTLVVGGTGF IGQF ITKA SLGF GYP 12 n reductase (LAR) TFLLVRPGPVSP SKAVIIKTFQDKGAKVIYGVINDKEC
MEKILKEYEIDVVISLVGGARLLDQLTLLEAIKSVKTIK
RFLP SEFGHDVDRTDPVEPGLTMYKEKRLVRRAVEEY
Desmodium GIPFTNICCNSIASWPYYDNCHP SQVPPPMDQFQIYGD
uncinatum GNTKAYF ID GNDIGKF TMKTIDDIRTLNKNVHFRP S SN
CY S INELA SLWEKKIGRTLPRF TVTADKLLAHAAENII
PE S IV S SF THDIF INGC QVNF SIDEHSDVEIDTLYPDEKF
Accession:
RSLDDCYEDFVPMVHDKIHAGKSGEIKIKDGKPLVQT
Q84V83.1 GTIEEINKD IKTLVET QPNEEIKKDMKALVEAVPIS AM
G
Anthocyanin Alf S SVAVPRVEILASSGIESIPKEYVRPQEELTTIGNIFD 13 dioxygenase (ANS) EEKKDEGPQVPTIDLRDIDSDDQQVRQRCRDELKKAA
VDWGVMHLVNHGIPDHLIDRVKKAGQAFFELPVEVK
EKYANDQASGNIQGYGSKLANNASGQLEWEDYYFHL
Car/ca papaya IFPEEKRDLAIWPNNPADYIEVT SEYARQLRRLV SKIL
GVLSLGLGLEEGRLEKEVGGLDELLLQMKINYYPTCP
QPELALGVEAHTDIS AL TF ILHNMVP GL QLF YEGKWV
Accession:
TAKCVPNSIVMHVGDTIEIL SNGKYKSILHRGLVNKEK
XP 021901846.1 VRISWAVFCEPPKEKIILKPLPETVSENEPPLFPPRTFAQ
HIQHKLFRKNQENLEAK
Anthocy ani di n-3 - MS QTT TNPHVAVLAFPF STHAAPLLAVVRRLAVAAPH 14 0-glycotransferase AVF SFF S T SE SNA SIFHD SMHTMQCNIK SYDVSDGVPE
(3 GT) GYVF T GRP QEGIDLFIVIRAAPE SFRQ GMVMAVAET GR
PVSCLVADAFIWFAADMAAEMGVAWLPFWTAGPNS
L S THVYIDEIREKIGV S GIQ GREDELLNF IP GM SKVRFR
Vitis labrusca DLQEGIVFGNLNSLF SRLLHRMGQVLPKATAVF IN SFE
ELDDSLTNDLKSKLKTYLNIGPFNLITPPPVVPNTTGCL
QWLKERKPT SVVYISF GTVT TPPPAELVALAEALEA SR

Accession: VPF IW SLRDKARMHLPEGFLEKTRGHGMVVPWAPQ A

GDQRLNGRMVEDVLEIGVRIEGGVF TK S GLM S CFD Q I
LSQEKGKKLRENLRALRETADRAVGPKGS STENFKTL
VDLVSKPKDV
Acetyl -C oA MVEHRSLPGHFLGGNSLESAPQGPVKDFVQAHEGHT 15 carboxylase (ACC) VI SKVL IANNGMAAMKEIRS VRKWAYETF GNERAIEF
TVMATPEDLKANAEYIRMADNFVEVPGGSNNNNYAN
VELIVDVAERTAVHAVWAGWGHASENPRLPEMLAKS
Mucor KHKCLFIGPPA S AMR SL GDKI S STIVAQ SAQVPTMGW
circinelloides SGDGITETEFDAAGHVIVPDNAYNEACVKTAEQGLKA
1006PhL AEKIGFPVMIKASEGGGGKGIRMVKDGSNFAQLFAQV
QGEIPGSPIFIMKLAGNARHLEVQLLADQYGNAISLFG
RD C SVQRRHQKIIEEAPVTIAKPDVFEQMEKAAVRLG
Accession:
KLVGYVSAGTVEYLYSHHDDQFYFLELNPRLQVEHPT
EPB 82652.1 TEMVSGVNLPAAQLQIAMGIPLHRIRDIRVLYGVQPNS
A SEIDF GFEHP T SL T SHRRP TPKGHVIACRITAENPDAG
FKP S SGIIVIQELNFRS STNVWGYF SVVSAGGLHEYAD S
QFGHIFAYGENRQQARKNMVIALKEL SIRADFRSTVE
YIIRLLETPDFEENTINTGWLDMLISKKLTAERPDTML
AVFCGAVTKAHMASLDCFQQYKQ SLEKGQVP SKGSL
KTVFTVDFIYEEVRYNFTVTQ S AP GIYTLYLNGTK TQV
GIRDL SD GGLLI S ID GK SHT TY SRDEVQATRMMVD GK
TCLLEKE SDP T QLR SP SP GKLVNLLVENGDHLNAGDA
YAEIEVMKMYMPLIATEDGHVQFIKQAGATLEAGDII
GIL SLDDP SRVKHALPFNGTVPAFGAPHITGDKPVQRF
NATKLTLQHILQGYDNQALVQTVVKDFADILNNPDLP
YSELNSVL SAL S GRIP QRLEASIHKLADE SKAANQEFP
AAQFEKLVEDFAREHITLQ SEATAYKNSVAPLS SIF AR
YRNGLTEHAYSNYVELMEAYYDVEILFNQQREEEVIL

SLRDQHKDDLDKVLAVTLSHAKVNIKNNVILMLLDLI
NPV S T GS ALDKYF TPILKRL SEIESRATQKVTLKAREL
LILCQLP SYEERQAQMYQILKNSVTESVYGGGSEYRTP
SYDAFKDLIDTKFNVFDVLPHF'FYHADPYIALAAIEVY
CRRSYHAYKILDVAYNLEHKPYVVAWKFLLQTAANG
ID SNKRIASYSDLTFLLNKTEEEPIRTGAMTACNSLAD
LQAELPRILTAFEEEPLPPMLQRNAAPKEERMENILNI
AVRADEDMDDTAFRTKICEMITANADVFRQAHLRRL
SVVVCRDNQWPDYYTFRERENYQEDETIRHIEPAMA
YQLELARLSNFDIKPCFIENRQMHVYYAVAKENP SD C
RFFIRALVRPGRVKS SMRTADYLISESDRLLTDILDTLE
IV SHEYKN SD CNHLFINF IP TF AIEADDVEHALKDF VD
RHGKRLWKLRVTGAEIRFNVQ SKKPDAPIIPMRF TVD
NV S GFILKVEVYQEVKTEK SGWILK SVNKIPGAMHM
QPL STPYPTKEWLQPRRYKAHLMGTTYVYDFPELFRQ
SVQNQWTQAIKRNPLLKQP SHLVEAKELVLDEDDVL
QEIDRAPGTNTVGMVAWIMTIRTPEYPSGRRIIAIANDI
TFKIGSF GVAEDQVFYKASELARALGIPRIYL S AN S GA
RIGLADELI S QFRAAWKDA SNP TAGFKYLYL TPAEYD
VLAQQGDAK SVLVEEIQDEGETRLRITDVIGHTDGLG
VENLKGSGLIAGAT SRAYDDIF TITLVT CRS VGIGAYL
VRLGQRTIQNEGQPIILTGAPALNKVLGREVYTSNLQL
GGTQIMYKNGVSHLTAENDLEGIAKIVQWL SF VPDVR
NAP V SMRL GADPIDRDIEYTPPK GP SDPRFFLAGK SEN
GKWLSGFFDQD SF VETL SGWARTVVVGRARLGGIPM
GVV SVETRTVENIVPADPAN SD STEQVFMEAGGVWFP
N SAYKTAQ AINDFNKGEQLPLMIF ANWRGF SGGQRD
MYNEVLKYGAQIVDALSNYKQPVFVYIIPNGELRGGA
WVVVDPTINKDMMEMYADNNARGGVLEPEGIVEIKY
RKPALLATMERLDATYASLKKQLAEEGKTDEEKAAL
KVQVEAREQELLPVYQQISIQFADLHDRAGRMKAKG

VIRKALDWRRARHYFYWRVRRRLCEEYTFRKIVTATS
AAPMPREQMLDLVKQWF TNDNETVNFEDADELV SE
WFEKRASVIDQRISKLKSDATKEQIVSLGNADQEAVIE
GF SQLIENL SEDARAEILRKLNSRF
Acetyl -C oA MSQTHKHAIPANIADRCLINPEQYETKYKQ SINDPDTF 16 synthase (AC S) WGEQGKILDWITPYQKVKNT SF AP GNV SIKWYED GT
LNLAANCLDRHLQENGDRTAIIWEGDDTSQ SKHIS YR
ELHRDVCRFANTLLDLGIKKGDVVAIYMPMVPEAAV
Salmonella AMLACARIGAVHSVIFGGF SPEAVAGRIIDS S SRL VITA
Ophimurium DEGVRAGRSIPLKKNVDDALKNPNVT SVEHVIVLKRT
GSDIDWQEGRDLWWRDLIEKASPEHQPEAMNAEDPL
FILYT S GS TGKPKGVLHTTGGYLVYAATTFKYVFDYH
Accession:
PGDIYWC TAD VGW VT GH S YLL YGPLAC GAT TLMFEG
NP 463140.1 VPNWPTPARMCQVVDKHQVNILYTAPTAIRALMAEG
DKAIEGTDRS SLRIL GS VGEPINPEAWEWYWKKIGKE
KCPVVDTWWQTETGGFMITPLPGAIELKAGSATRPFF
GVQPALVDNEGHPQEGATEGNLVITDSWPGQARTLF
GDHERFEQTYF STFKNIVIYF SGDGARRDEDGYYWITG
RVDDVLNV S GHRL GTAEIE S AL VAHPKIAEAAVVGIP
HAIKGQAIYAYVTLNHGEEP SPELYAEVRNWVRKEIG
PL ATPDVLHW TD SLPK TR S GKIMRRILRKIAAGD T SNL
GD T S TL ADP GVVEKLLEEKQ AIAMP S
Mal onyl-C oA MS SLFPALSPAPTGAPADRPALRFGERSLTYAELAAA 17 synthase (matB) AGATAGRIGGAGRVAVWATPAMETGVAVVAALLAG
VAAVPLNPKSGDKELAHIL SD SAP SL VLAPPDAELPP A
LGALERVDVDVRARGAVPED GADD GDPALVVYT SGT
Streptomyces TGPPKGAVIPRRALATTLDALADAWQWTGEDVLVQG
coelicolor LPLFHVHGLVLGILGPLRRGGSVRHLGRF STEGAAREL
ND GATMLF GVP TMYHRIAETLPADPELAKALAGARL
LV S GS AALPVHDHERIAAATGRRVIERYGMTETLMNT

Accession: SVRADGEPRAGTVGVPLPGVELRLVEEDGTPIAALDG

DMAVRDPDGYVRIVGRKATDLIKSGGYKIGAGEIENA
LLEHPEVREAAVTGEPDPDLGERIVAWIVPADPAAPP
ALGTLADHVAARLAPHKRPRVVRYLDAVPRNDMGKI
MKRALNRD
Malonate MSPELISILVLVVVFVIATTRSVNMGALAFAAAFGVGT 18 transporter (matC) LVADLDADGIFAGFPGDLFVVLVGVTYLFAIARANGT
TDWLVHAAVRLVRGRVALIPWVMFALTGALTAIGAV
SPAAVAIVAPVALSFATRYSISPLLMGTMVVHGAQAG
Streptomyces GFSPISIYGSIVNGIVEREKLPGSEIGLFLASLVANLLIA
coelicolor AVLFAVLGGRKLWARGAVTPEGDGAPGKAGTGTTGS
GSDTGTGTGTGTGTSAGTGGTAPTAVAVRSDRETGG
AEGTGVRLTPARVATLVALVALVVAVLGFDLDAGLT
Accession:
AVTLAVVLSTAWPDDSRRAVGEIAWSTVLLICGVLTY
NP 626686.1 VGVLEEMGTITWAGEGVGGIGVPLLAAVLLCYIGAIV
SAFASSVGIIVIGALIPLAVPFLAQGEIGAVGMVAALAV
SATVVDVSPFSTNGALVLAAAPDVDRDRFFRQLMVY
GGIVVAAVPALAWLVLVVPGFG
Malonate CoA- MVKKRLWDKQRTRRQEKLNLAQQKGFAKQVEHARA 19 transferase (MdcA) IELLETVIASGDRVCLEGNNQKQADFLSKCLSQCNPD
AVNDLHIVQSVLALPSHIDVFEKGIASKVDFSFAGPQS
LRLAQLVQQQKISIGSIHTYLELYGRYFIDLTPNICLITA
Acinetobacter HAADREGNLYTGPNTEDTPAIVEATAFKSGIVIAQVNE
calcoaceticus IVDKLPRVDVPADWVDFYIESPKHNYIEPLFTRDPAQI
TEVQILMAMMVIKGIYAPYQVQRLNHGIGFDTAAIEL
LLPTYAASLGLKGQICTNWALNPHPTLIPAIESGFVDS
Accession:
VHSFGSEVGMEDYIKERPDVFFTGSDGSMRSNRAFSQ
AAB97627.1 TAGLYACDSFIGSTLQIELQGNSSTATVDRISGFGGAP
NMGSDPHGRRHASYAYTKAGREATDGKLIKGRKLVV

QTVETYREHMHPVFVEELDAWQLQDKMDSELPPIIVII
YGEDVTHIVTEEGIANLLLCRTDEEREQAIRGVAGYTP
VGLKRDAAKVEELRQRGIIQRPEDLGIDPTQVSRDLLA
AKSVKDLVKWSGGLYSPPSRFRNW
Pantothenate kinase MILELDCGNSLIKWRVIEGAARSVAGGLAESDDALVE 20 (CoaX) QLTSQQALPVRACRLVSVRSEQETSQLVARLEQLFPV
SALVASSGKQLAGVRNGYLDYQRLGLDRWLALVAA
Pseudomonas HHLAKKACLVIDLGTAVTSDLVAADGVHLGGYICPG
aeruginosa MTLMRSQLRTHTRRIRYDDAEARRALASLQPGQATA
Accession: EAVERGCLLMLRGFVREQYAMACELLGPDCEIFLTGG
Q9HWC1.1 DAELVRDELAGARIMPDLVFVGLALACPIE
glutamyl-tRNA MTKKLLALGINHKTAPVSLRERVTFSPDTLDQALDSL 21 reductase (hemAm) LAQPMVQGGVVLSTCNRTELYLSVEEQDNLQEALIR
WLCDYHNLNEDDLRNSLYWHQDNDAVSHLMRVASG
LDSLVLGEPQILGQVKKAFADSQKGHLNASALRRMF
Salmonella QKSFSVAKRVRTETDIGASAVSVAFAACTLARQIFESL
Ophimurium STVTVLLVGAGETIELVARHLREHKVQKMIIANRTRE
RAQALADEVGAEVISLSDIDARLQDADIIISSTASPLPII
GKGMVERALKSRRNQPMLLVDIAVPRDVEPEVGKLA
Accession:
NAYLYSVDDLQSIISHNLAQRQAAAVEAETIVEQEASE
AAA88610.1 FMAWLRAQGASETIREYRSQSEQIRDELTTKALSALQ
QGGDAQAILQDLAWKLTNRLIHAPTKSLQQAARDGD
DERLNILRDSLGLE
5-aminolevulinic MDYNLALDKAIQKLHDEGRYRTFIDIEREKGAFPKAQ 22 acid synthase WNRPDGGKQDITVWCGNDYLGMGQHPVVLAAMHE
(ALAS) ALEAVGAGSGGTRNISGTTAYHRRLEAEIADLHGKEA
ALVFSSAYIANDATLSTLRLLFPGLIIYSDSLNHASMIE
GIKRNAGPKRIFRHNDVAHLRELIAADDPAAPKLIAFE
SVYSMDGDFGPIKEICDIADEFGALTYIDEVHAVGMY

Rhodobacter GPRGAGVAERDGLMHRIDIFNGTLAKAYGVF GGYIA
capsulatus A S AKMVD AVR S YAP GF IF S T SLPP AIAAGAQ A S IAF LK
TAEGQKLRDAQQMHAKVLKMRLKALGMPIIDHGSHI
VPVVIGDPVHTKAVSDMLL SDYGVYVQPINFPTVPRG
Accession: TERLRF TP SPVHDLK Q ID GL VHAMDLLWARC A

Tyrosine ammonia- MTLQSQTAKDCLALDGALTLVQCEAIATHRSRISVTP 23 lyase (TAL) ALRERCARAHARLEHAIAEQRHIYGITTGF GPLANRLI
GAD Q GAELQ QNLIYHLAT GVGPKL SWAEARALMLAR
LNSILQGASGASPETIDRIVAVLNAGFAPEVPAQGTVG
Rhodobacter A S GDL TPL AHMVLAL Q GRGRMIDP SGRVQEAGAVM
capsulatus SB 1003 DRLCGGPLTLAARDGLALVNGT SAMTAIAALTGVEA
ARAIDAALRHSAVLMEVL SGHAEAWHPAFAELRPHP
GQLRATERLAQALDGAGRVCRTLTAARRLTAADLRP
Accession:
EDHPAQDAYSLRVVPQLVGAVWDTLDWHDRVVTCE
ADE84832.1 LNSVTDNPIFPEGCAVPALHGGNFMGVHVALASDAL
NAALVTLAGLVERQIARLTDEKLNKGLPAFLHGGQA
GLQ S GF MGAQ VTAT ALL AEMRANATPV S VQ SL STNG
ANQDVVSMGTIAARRARAQLLPL S QIQAILAL AL AQ A
MDLLDDPEGQAGW SL TARDLRDRIRAV SP GLRADRP
LAGHIEAVAQGLRHP SAAADPPA
Tyrosine ammonia- MI TE TNVAKP A S TKVNINGD AAKAAP VEPF AT YAH S Q 24 lyase (TAL) ATKTVVIDGHNMKVGDVVAVARHGAKVELAASVAG
PVQ A SVDFKESKKHT SIYGVTTGF GGS AD TRT SD TEA
LQISLLEHQLCGYLPTDPTYEGMLLAAMPIPIVRGAM
Trichosporon AVRVNSCVRGHSGVRLEVLQ SF ADF INIGL VP C VPLR
cutaneum GTI S A SGDL SPL S YIAGAIC GHPD VKVF D T AA SPP T VL T
APEAIAKYKLKTVRLASKEGLGLVNGTAVSAAAGAL
ALYD AECL AMM S Q TNT AL TVEALD GHVGSF APF IQEI
Accession:
RPHVGQIEAAKNIRHMLSNSKLAVHEEPELLADQDAG

DNPL ID VE GGMF HHGGNF QAMAVT SAMD S TRIVLQN
LGKL SF AQ VTELINCEMNHGLP SNLAGSEP STNYHCK
GLDIHCGAYCAELGFLANPMSNHVQ S TEMHNQ SVNS
MAFASARKTMEANEVLSLLLGSQMYCATQALDLRV
MEVKFKMAIVKLLNDTLTKHF S TF L TPEQL AKLNT T A
AITLYKRLNQTP SWD S APRF ED AAKHL VGC IMD ALM
VNDDITDLTNLPKWKKEFAKDAGDLYRSILTATTADG
RNDLEPAEYLGQTRAVYEAIRSDLGVKVRRGDVAEG
K SGK SIGSNVARIVEAM RD GRLMGAV SKMF F
Tyrosine ammonia- MNTINEYL SLEEFEAIIF GNQKVTI SD VVVNRVNE SFNF 25 lyase (TAL) LKEF SGNKVIYGVNTGF GPMAQ YRIKE SD Q IQL Q YNLI
RSHS S GT GKPL SP VC AKAAILARLNTL SLGNSGVHP SV
INLMSELINKDITPLIFEHGGVGASGDLVQL SHL AL VLI
Flavobacterium GEGEVFYKGERRPTPEVFEIEGLKPIQVEIREGLALING
johnsoniae T SVMTGIGVVNVYHAKKLLDW SLK S S C AINELVQ AY
DDHF SAELNQTKRHKGQQEIALKMRQNL SD S TLIRKR
EDHLYSGENTEEIFKEKVQEYYSLRCVPQILGPVLETI
Accession:
NNVASILEDEFNSANDNPIIDVKNQHVYHGGNFHGDY

NLGTLGFNF GMQGVQF TAT S TTAESQML SNPMYVHSI
PNNNDNQDIVSMGTNSAVIT SKVIENAFEVLAIEMITIV
Q AID YL GQKDKI S S V SKKWYDEIRNIIP TF KED Q VMYP
FVQKVKDHLINN
Tyrosine ammonia- MSTTLILTGEGLGIDDVVRVARHQDRVELTTDPAILA 26 lyase (TAL) QIEASCAYINQAVKEHQPVYGVTTGF GGMANVIISPEE
AAELQNNAIWYHKTGAGKLLPF TDVRAAMLLRANSH
MRGASGIRLEIIQRMVTFLNANVTPHVREF GS IGA S GD
LVPLI S ITGALLGTD QAFMVDFNGETLD C I S ALERLGL
PRLRLQPKEGLAMMNGT SVMTGIAANCVHDARILLA

Herpetosiphon LALEAHALMIQGLQGTNQ SFHPFIHRHKPHTGQVWA
aurantiacus DSM ADHMLELLQGSQLSRNELDGSHDYRDGDLIQDRYSL

A SYHGGNFL GQYIGVGMD QLRYYMGLMAKHLDVQI
ALLVSPQFNNGLPASLVGNIQRKVNMGLKGLQLTANS
Accession: IMPILTFLGNSLADRFPTHAEQFNQNINSQGFGSANLA
ABX04526.1 RQTIQTLQQYIAITLMFGVQAVDLRTHKLAGHYNAAE
LL SPLTAKIYHAVRSIVKHPP SPERPYIWNDDEQVLEA
HISALAHDIANDGSLVSAVEQTL SGLRSIILFR
Phenylalanine MHDDNT SPYCIGQLGNGAVHGADPLNWAKTAKAME 27 ammonia-lyase CSHLEEIKRMVDTYQNATQVMIEGATLTVPQVAAIAR
(PAL) RPEVHVVLDAANARSRVDES SNWVLDRIMGGGDIYG
VT T GF GAT SHRRT Q Q GVELQRELIRFLNAGVL SKGNS
LP SET ARAAMLVRTNTLMQ GY S GIRWEILHAMEKLL
Physcomitrella NAHVTPKLPLRGTITASGDLVPLSYIAGLLTGRPNSKA
patens VTEDGREVSALEALRIAGVEKPFELAPKEGLALVNGT
AVGSALASTVCYDANEVIVLLAEVL S ALF CEVMQ GKP
EF ADPLTHKLKHHP GQMEAAAVMEWVLD GS SFMKA
Accession:
AAKFNETDPLRKPKQDRYALRT SPQWLGPQVEVIRNA
XP 001758374.1 THAIEREINSVNDNPIIDAARGIALHGGNFQGTPIGVSM
DNMRL SLAAIAKLMFAQF SELVNDYYNNGLP SNLSG
GPNP SLDYGMKGAEIAMASYL SEINYLANPVTTHVQ S
AEQHNQDVNSLGLVSARKTEEAMEILKLMSATFLVG
LC QAIDLRHVEETMQ S AVKQVVT QVAKKTLFMG SD G
SLLP SRFCEKELLMVVDRQPVF SYIDDST SD SYPLMEK
LRGVLVSRALKSADKETSNAVFRQIPVFEAELKLQL SR
VVPAVREAYDTKGL SLVPNRIQDCRTYPLYKLVRGDL
K TQLL S GQRTV SP GQEIEKVFNAI SAGQLVAPLLECVQ
GWTGTPGPF SARASC

Phenylalanine MIETNHKDNFLID GENKNLEINDII S I SK GEKNIIF TNEL 28 ammonia-lyase LEFLQKGRDQLENKLKENVAIYGINTGFGGNGDLIIPF
(PAL) DKLDYHQ SNLLDFL T C GT GDFFND Q YVRGIQF IIIIAL S
RGW S GVRPMVIQ TL AKHLNK GIIP Q VPMHGS VGA S G
DLVPL SYIANVLCGKGMVKYNEKLMNASDALKIT SHE
Dictyostelium PLVLKSKEGLALVNGTRVMS SVSCISINKFETIFKAAIG
discoideum AX4 SIALAVEGLLASKDHYDMRIHNLKNHPGQILIAQILNK
YFNT SDNNTK S SNITFNQ SENVQKLDKSVQEVYSLRC
APQIL GII SENT SNAKIVIKREIL SVNDNPLIDPYYGDVL
Accession:
SGGNFMGNHIARIMDGIKLDISLVANHLHSLVALM MH
XP 644510.1 SEF SKGLPNSLSPNPGIYQGYKGMQISQTSLVVWLRQE
AAPACIH SL T TEQFNQDIV SL GLH SANGAA SMLIKL CD
IV SMTL IIAF Q AI SLRMK S IENFKLPNKVQKLY S SIIKIIPI
LENDRRTDIDVREITNAILQDKLDFINLNL
Phenylalanine MSQVALFEQELMLHGKHTLLLNGNDLTITDVAQMAK 29 ammonia-lyase GTFEAFTFHISEEANKRIEECNELKHEIMNQHNPIYGV
(PAL) TTGFGD SVHRQISGEKAWDLQRNLIRFL SCGVGPVAD
EAVARATMLIRTNCLVKGNSAVRLEVIHQLIAYMERG
ITPIIPERGSVGA SGDL VPL SYLASILVGEGKVL YK GEE
Brevi bacillus REVAEAL GAEGLEPL TLEAKEGLALVNGT SFM S AF AC
laterosporus LIIIG LAYADAEEIAF IADIC TAMA SEALL GNRGHF Y SF IHEQ

SKAYLELTQ SIQDRYSIRCAPHVTGVLYDTLDWVKK
WLEVEINSTNDNPIFDVETRDVYNGGNEYGGHVVQA
Accession:
MD SLKVAVANIADLLDRQLQLVVDEKFNKDLTPNLIP
WP 00333 7219.1 RENNDNYEIGLHHGFKGMQIAS S AL TAEALKM S GPV S
VF SRSTEAHNQDKVSMGTIS SRDARTIVELTQHVAAIH
LIALCQALDLRD SKKM SP Q T TKIYNMIRK Q VPF VERD
RALDGDIEKVVQLIRSGNLKKEIHDQNVND

Cinnamate-4- MDLLLMEKTLLGLFVAVVVAITVSKLRGKKFKLPPGP 30 hydroxylase (C4H) IPVPVFGNWLQVGDDLNHRNLTEMAKKFGEVFMLR
MGQRNLVVVSSPDLAKEVLHTQGVEFGSRTRNVVFDI
FTGKGQDMVFTVYGEHWRKMRRIMTVPFFTNKVVQ
Rubus sp. SSL-2007 QYRYGWESEAAAVVEDVKKHPEAATNGMVLRRRLQ
LMMYNNMYRIMFDRRFESEDDPLFVKLKGLNGERSR
LAQSFEYNYGDFIPVLRPFLRGYLKICKEVKEKRIQLF
Accession:
KDYFVDERKKLSSTQATTNEGLKCAIDHILDAQQKGE
ABX74781.1 INEDNVLYIVENINVAAIETTLWSIEWGIAELVNHPEIQ
KKLRDELDTVLGRGVQITEPEIQKLPYLQAVVKETLR
LRMAIPLLVPHMNLHDAKLGGFDIPAESKILVNAWWL
ANNPAHWKKPEEFRPERFLEEESKVEANGNDFRYLPF
GVGRRSCPGIILALPILGITLGRLVQNFELLPPPGQTQL
DTTEKGGQFSLHILKHSPIVMKPRT
Cinnamate-4- MDLLLLEKTLIGLFIAIVVAIIVSKLRGKKFKLPPGPIPV 31 hydroxylase (C4H) PVFGNWLQVGDDLNEIRNLTDMAKKFGDVFMLRMG
QRNLVVVSSPDLAKEVLHTQGVEFGSRTRNVVFDIFT
GKGQDMVFTVYGEHWRKMRRIIVITVPFFTNKVVQQY
Fragaria vesca RHGWEAEAAAVVEDVKKHPEAATSGMVLRRRLQLM
MYNNMYRIMFDRRFESEEDPLFVKLKGLNGERSRLA
QSFEYNYGDFIPVLRPFLRGYLKICKEVKEKRIQLFKD
Accession:
YFVDERKKLASTQVTTNEGLKCAIDHILDAQQKGEIN
XP 004294725.1 EDNVLYIVENINVAAIETTLWSIEWGIAELVNHPEIQK
KLRDELDTVLGHGVQVTEPELHKLPYLQAVVKETLR
LRMAIPLLVPHMNLHDAKLGGFDIPAESKILVNAWWL
ANNPAHWKKPEEFRPERFLEEESKVEANGNDFRYLPF
GVGRRSCPGIILALPILGVTLGRLVQNFEMLPPPGQTQ
LDTTEKGGQFSLHILKHSTIVMKPRA
Cinnamate-4- MDLLLLEKTLIGLFFAILIAIIVSKLRSKRFKLPPGPIPVP 32 hydroxylase (C4H) VFGNWLQVGDDLNEIRNLTEYAKKFGDVFLLRMGQR

NLVVVSSPELAKEVLHTQGVEFGSRTRNVVFDIFTGK
GQDMVFTVYGEHWRKMRRIIVITVPFFTNKVVQQYRG
Solanum tuberosum GWESEAASVVEDVKKNPESATNGIVLRKRLQLMMYN
NMFRIMFDRRFESEDDPLFVKLRALNGERSRLAQSFE
Accession: YNYGDFIPILRPFLRGYLKICKEVKEKRLKLFKDYFVD
ERKKLANTKSMDSNALKCAIDHILEAQQKGEINEDNV
ABC69046.1 LYIVENINVAAIETTLWSIEWGIAELVNHPHIQKKLRD
EIDTVLGPGMQVTEPDMPKLPYLQAVIKETLRLRMAI
PLLVPHMNLHDAKLAGYDIPAESKILVNAWWLANNP
AHWKKPEEFRPERFFEEEKHVEANGNDFRFLPFGVGR
RSCPGIILALPILGITLGRLVQNFEMLPPPGQSKLDTSE
KGGQFSLHILKHSTIVMKPRSF
4-coumarate-CoA MGDCAAPKQEBFRSKLPDIYIPKHLPLHSYCFENISKV 33 ligase (4CL) SDRACLINGATGETFSYAQVELISRRVASGLNKLGIHQ
GDTMMILLPNTPEYFFAFLGASYRGAVSTMANPFF TS
PEVIKQLKASQAKLIITQACYVEKVKEYAAENNITVVC
Daucus carota IDEAPRDCLHFTTLMEADEAEMPEVAIDSDDVVALPY
SSGTTGLPKGVMLTHKGLVTSVAQRVDGENPNLYIHS
EDVMICILPLFHIYSLNAVLCCGLRAGATILIMQKFDIV
Accession:
PFLELIQKYKVTIGPFVPPIVLAIAKSPVVDNYDLSSVR
AIT52344.1 TVMSGAAPLGKELEDAVRAKFPNAKLGQGYGMTEA
GPVLAMCLAFAKEPYEIKSGACGTVVRNAEMKIVDPE
THASLPRNQSGEICIRGDQIMKGYLNDPESTKTTIDEE
GWLHTGDIGFIDEDDELFIVDRLKEIIKYKGFQVAPAEI
EALLLTHPTISDAAVVPMIDEKAGEVPVAFVVRLNGS
TTTEEEIKQFVSKQVVFYKRVFRVFFVDAIPKSPSGKIL
RKELRARIASGDLPK
4-coumarate-CoA MEPTTKSKDIIFRSKLPDIYIPKHLPLHTYCFENISRFGS 34 ligase (4CL) RPCLINGSTGEILTYDQVELASRRVGSGLHRLGIRQGD
TIMLLLPNSPEFVLAFLGASHIGAVSTMANPFFTPAEV

VKQAAASRAKLIVTQACHVDKVRDYAAEHGVKVVC
VDGAPPEECLPFSEVASGDEAELPAVKISPDDVVALPY
Striga as/at/ca SSGTTGLPKGVMLTHKGLVTSVAQQVDGENPNLYIES
DDVIIVICVLPLFHIYSLNSIMLCGLRVGAAILIIVIQKFEIV
Accession: PFLELIQRYRVTIGPFVPPIVLAIEKSPVVEKYDLSSVRT
VMSGAAPLGRELEDAVRLKFPNAKLGQGYGMTEAGP
GER48539.1 VLAMCLAFAKEPFEIKSGACGTVVRNAEMKIVDTETG
ASLGRNQPGEICIRGDQIMKGYLNDPESTERTIDKEGW
LHTGDIGFIDDDDELFIVDRLKEIIKYKGFQVAPAELEA
LLLNHPNISDAAVVSMKDEQAGEVPVAYVVKSNGSTI
TEDEIKQFVSKQVIFYKRINRVFFIDAIPKSPSGKILRKD
LRARLAAGVPN
4-coumarate-CoA MPMENEAKQGDIIFRSKLPDIYIPNHLSLHSYCFENISE 35 ligase (4CL) FSSRPCLINGANNQIYTYADVELNSRKVAAGLHKQFGI
QQKDTIMILLPNSPEFVFAFLGASYLGAISTMANPLFTP
AEVVKQVKASNAEIIVTQACHVNKVKDYALENDVKI
Capsicum annuum VCIDSAPEGCVHFSELIQADEHDIPEVQIKPDDVVALP
YSSGTTGLPKGVMLTHKGLVTSVAQQVDGENPNLYI
HSEDVMLCVLPLFHIYSLNSVLLCGLRVGAAILIMQKF
Accession:
DIVPFLELIQNYKVTIGPFVPPIVLAIAKSPMVDNYDLS
KAF3620179.1 SVRTVMSGAAPLGKELEDTVRAKFPNAKLGQGYGMT
EAGPVLAMCLAFAKEPFEIKSGACGTVVRNAEMKIVD
PDTGNSLHRNQSGEICIRGDQIMKGYLNDPEATAGTID
KEGWLHTGDIGYIDNDDELFIVDRLKELIKYKGFQVA
PAELEALLLNHPNISDAAVVPMKDEQAGEVPVAFVVR
SNGSTITEDEVKEFISKQVIFYKRIKRVFFVDAVPKSPS
GKILRKDLRAKLAAGFPN
4-coumarate-CoA MDTKTTQQEIIFRSKLPDIYIPKQLPLHSYCFENISQFSS 36 ligase (4CL) KPCLINGSTGKVYTYSDVELTSRKVAAGFHNLGIQQR
DTIMLLLPNCPEFVFAFLGASYLGAIITMANPFFTPAET

IKQAKASNSKLIITQ S SYT SKVLDYS SENNVKIICID SPP
DGCLHF SELIQ SNETQLPEVEID SNEVVALPYS S GT T GL
Camellia sinensis PKGVMLTHKGLVTSVAQQVDGENPNLYIHSEDMMM
CVLPLFHIYSLNSVLLCGLRVGAAILIMQKFEIGSFLKL
Accession: IQRYKVTIGPFVPPIVLAIAKSEVVDDYDL STIRTMMS
GAAPLGKELEDAVRAKFPHAKLGQGYGMTEAGPVLA
ASU87409.1 MCLAFAKKPFEIK SGACGTVVRNAEMKIVDPDAGF SL
PRNQPGEICIRGDQIMKGYLNDPEATERTIDKQGWLH
TGDIGYIDDDDELFIVDRLKELIKYKGFQVAPAELEAL
LLNHPTISDAAVVPMKDESAGEVPVAFVVRTNGFEVT
ENEIKKYISEQVVFYKKINRVYFVDAIPKAPSGKILRK
DLRARLAAGIPS
Chal cone syntha se MVTVEEYRKAQRAEGPATVMAIGTATP SNC VD Q STY 37 (CHS) PDYYFRITNSEHKTELKEKFKRMCEKSMIKTRYMHLT
EEILKENPNMCAYMAPSLDARQDIVVVEVPKLGKEA
AQKAIKEWGQPKSKITHLVFCTTSGVDMPGCDYQLA
Capsicum annuum KLLGLRPSVKRLMMYQQGCFAGGTVLRLAKDLAEN
NKGARVLVVC SEITAVTFRGP SE SHLD SL VGQ ALF GD
GAAAIIIVIGSDPIPGVERPLF QLVSAAQTLLPD SEGAID
Accession:
GHLREVGLTFHLLKDVPGLISKNIEKSLVEAF QPLGI SD
XP 016566084.1 WNSLFWIAHPGGPAILDQVELKLGLKPEKLKATREVL
SNYGNIVIS S ACVLFILDEMRKA S TKEGL GT SGEGLEW
GVLF GF GP GLT VETVVLH S VAT
Chal cone syntha se MVTVEEVRKAQRAEGPATVLAIGTATPPNCIDQ STYP 38 (CHS) DYYFRITKSEHKAELKEKF QRMCDKSMIKKRYMYLT
EEILKENP SMCEYMAP SLDARQDMVVVEIPKLGKEAA
TKAIKEWGQPKSKITHLVFC TT SGVDMPGADYQLTKL
Rosa chinensis LGLRPSVKRLMMYQQGCFAGGTVLRLAKDLAENNK
GARVLVVC SEITAVTFRGP SD THLD SLVGQ ALF GD GA
AAIIVGSDPLPEVEKPLFELVSAAQTILPDSDGAIDGHL

Accession: REVGLTFHLLKDVPGLISKNIEKSLNEAFKPLNITDWN
AEC13058.1 SLFWIAHPGGPAILDQVEAKLGLKPEKLEATRHILSEY
GNIVIS SACVLFILDEVRRKSAANGHKTTGEGLEWGVL
FGFGPGLTVETVVLHSVAA
Chal cone syntha se MSMTPSVHEIRKAQRSEGPATVL SIGTATP TNF VP QAD 39 (CHS) YPDYYFRITNSDHMTDLKDKFKRIVICEKSMITKRHMY
LTEEILKENPKMCEYMAPSLDARQDIVVVEVPKLGKE
AAAKAIKEWGQPKSKITHLIFCTTSGVDMPGADYQLT
Moms alba var. KLLGLRPSVKRFMMYQQGCFAGGTVLRLAKDLAENN
mull/can/is KGARVLVVC SETT AVTFRGP SHTHLD SLVGQ ALF GDG
AAAVILGADPDT S VERP IFEL V S AAQ T ILPD SEGAIDGH
LREVGLTFHLLKDVPGLISKNIEKSLVEAFTPIGISDWN
Accession:
SIFWIAHPGGPAILDQVEAKLGLKQEKL SATRHVL SEY
AHL83549.1 GNIVIS SACVLFILDEVRKKSVEEGKATTGEGLEWGVLF
GFGPGLTVETIVLHSLPAV
Chalcone synthase MAPPAMEEIRRAQRAEGPATVLAIGASTPPNALYQAD 40 (CHS) YPDYYFRITKSEHLTELKEKFKQMCDKSMIRKRYMYL
TEEILKENPNICAFMAPSLDARQDIVVTEVPKLAKEAS
ARAIKEWGQPKSRITHLIFCTTSGVDMPGADYQLTRL
Dendrobium LGLRPSVNRIMLYQQGCFAGGTVLRLAKDLAENNAG
catenatum ARVLVVC SETT AVTF RGP SE SHLD SL VGQ ALF GDGAA
AIIVGSDPDLTTERPLFQLVSASQTILPESEGAIDGHLRE
MGLTFHLLKDVPGLISKNIQKSLVETFKPLGIHDWNSI
Accession:
FWIAHPGGPAILDQVEIKLGLKEEKLAS SRNVLAEYG
ALE71934.1 NMSSACVLFILDEMRRRSAEAGQATTGEGLEWGVLF
GFGPGLTVETVVLRSVPIAGAV

Chalcone isomerase MSAITAIHVENIEFPAVITSPVTGKSYFLGGAGERGLTI 41 (CHI) EGNFIKFTAIGVYLEDVAVASLATKWKGKSSEELLET
LDFYRDIISGPFEKLIRGSKIRELSGPEYSRKVTENCVA
Trifohum pratense HLKSVGTYGDAEVEAMEKFVEAFKPINFPPGASVFYR
Accession: QSPDGILGVSISIHFFP
PNX83855.1 Chalcone isomerase MAAASLTAVQVENLEFPAVVTSPATGKTYFLGGAGV 42 (CHI) RGLTIEGNFIKFTGIGVYLEDQAVASLATKWKGKS SEE
LVESLDFFRDIISGPFEKLIRGSKIRQLSGPEYSKKVME
Abrus precatorius NCVAHMKSVGTYGDAEAAGIEEFAQAFKPVNFPPGA
Accession: SVFYRQSPDGVLGLSFSQDATIPEEEAAVIKNKPVSAA
XP 027366189.1 VLETMIGEHAVSPDLKRSLAARLPAVLSHGVFKIGN
Chalcone isomerase MAAEPSITAIQFENLVFPAVVTPPGSSKSYFLAGAGER 43 (CHI) GLTIDGKFIKFTGIGVYLEDKAVPSLAGKWKDKSSQQ
LLQTLHFYRDIISGPFEKLIRGSKILALSGVEYSRKVME
Arachis duranensis NCVAHMKSVGTYGDAEAEAIQQFAEAFKNVNFKPGA
Accession: SVFYRQSPLGHLGLSFSQDGNIPEKEAAVIENKPLSSA
XP 015942246.1 VLETMIGEHAVSPDLKCSLAARLPAVLQQGIIVTPPQH
N
Chalcone isomerase MGPSPSVTELQVENVTFPPSVKPPGSTKTLFLGGAGER 44 (CHI) GLEIQGKFIKFTAIGVYLEGDAVASLAVKWKGKSKEE
LTD SVEFFRDIVTGPFEKFTQVTTILPLTGQQYSEKVSE
Cephalotus NCVAFWKSVGIYTDAEAKAIEKFIEVFKEETFPPGSSIL
folhcularis FTQSPNGALTIAFSKDGVIPEVGKAVIENKLLAEGLLE
Accession: SIIGKHGVSPVAKQCLATRLSELL
GAV77263.1 Flavanone 3- MGSASETVCVTGAAGFIGSWLVMRLIQNGYKVRATV 45 hy droxyl as e (F3H) RDPANMKKVKHLLELPNAKTNL SLWKADLAEEGSFD
EAIKGCTGVFHVATPMDFESKDPENEVIKPTINGLIDI
Abrus precatorius MKACMKAKTVRRLVFTSSAGTVDVTEHPKPLFDESC
Accession: W SD VQF CRRVRM T GWMYF V SK TL AEQEAWKF AKEN
XP 027329642.1 NIDF I S VIPPLVVGPF L VP TMPP SL IT AL SL IT GNE SHYAI
IKQGQFVEILDDLCLAHIFLFQHPKAQGRYICC SHEAT I
HDIASLLNQKYPEFNVPTKFKNIPDQLEIIRF S SKKITDL
GFKF KY SLEDMF T GAVE T CKEKRLL SET AEI S GT T QK
Flavanone 3- MKD S VA S ATA S AP GTVCVT GAAGF IG SWLVMRLLER 46 hy droxyl as e (F3H) GYIVRATVRDPANLKKVKHLLDLPKADTNLTLWKAD
LNEEGSFDEAIEGC SGVFHVATPMDFESKDPENEVIKP
Camellia sinensis TINGVLSIIRSCTKAKTVKRLVFTSSAGTVNVQEHQQP
Accession: VFDENNW SDLHF INKKKMTGWMYF V SKTLAEKAAW
05.1AAT665 EAAKENNIDF I S IIP TLVGGPF IMP TF PP SL I TAL SPITRN
EGHY S IIK Q GQF VHLDDL CE SHIFL YERP Q AEGRYIC S S
HDATIHDLAKLMREKWPEYNVPTEFKGIDKDLPVVSF
S SKKLIGMGFEFKY SLEDMFRGAID TCREKGLLPH SF A
ENPVNGNKV
Flavanone 3- MVDMKDDD SPATVCVTGAAGFIGSWLIMRLLQQGYI 47 hy droxyl as e (F3H) VRATVRDPANIVIKKVKHLQELEKADKNLTLWKADLT
EEGSF DEAIK GC SGVFHVATPMDFESKDPENEVIKPTI
Nyssa sinensis NGVLSIVRSCVKAKTVKRLVFTSSAGTVNLQEHQQLV
Accession: YDENNWSDLDLIYAKKMTGWMYFVSKILAEKAAWE
KAA8531902 .1 ATKENNIDF I SIIP TLVVGPF I TP TFPP SLITAL SLITGNEA
HY SIIKQ GQF VHLDDLCEAHIFLYEQPKAEGRYIC S SH
DATIYDLAKMIREKWPEYNVP TELKGIEKDLQ TV SF S S
KKLIGMGFEFKYSLEDMYKGAIDTCREKGLLPYSTHE
TPANANANANANVKKNQNENTEI

Flavanone 3- MA SE SE S VC VT GA S GFVG SWLVMRLLDRGYTVRATV 48 hy droxyl as e (F3H) RDPANKKKVKHLLDLPKAATHLTLWKADLAEEGSFD
EAIKGCTGVFHVATPMDFESKDPENEVIKPTINGVLDI
MKACLKAKTVRRLVF TA S AGS VNVEETQKPVYDE SN
Rosa chinensis W SD VEF CRRVKMTGWMYF A SKTLAEQEAWKF AKEN
NIDFITIIPTLVIGPFLMPAMPP SLITGL SPLT GNE SHY S II
KQGQFIHLDDLCQ SHIYLYEHPKAEGRYICS SHDATIH
Accession:
EIAKLLREKYPEYNVPTTFKGIEENLPKVHF S SKKLLE
)CP 024167119.1 TGFEFKYSLEDMFVGAVDACKAKGLLPPPTERVEKQE
VDES SVVGVKVTG
Flavonoid 3' MSPLILYSIALAIFLYCLRTLLKRHPHRLPPGPRPWPIIG 49 hy droxyl as e (F3 'H) NLPHMGQMPHHSLAAMARTYGPLMHLRLGFVDVIV
AA S A SVA S QLLKTHDANF S SRPHNSGAKYIAYNYQDL
VFAPYGPRWRMLRKIS SVHLF SGKALDDYRHVRQEE
Cephalotus VAVLIRALARAESKQAVNLGQLLNVCTANALGRVML
folhcularis GRRVF GD GS GV SDPMAEEFK SMVVEVMALAGVFNIG
DFIPALDWLDLQGVAAKMKNLHKRFDTFLTGLLEEH
KKMLVGDGGSEKHKDLL STLISLKDSADDEGLKLTDT
Accession:
EIKALLLNMFTAGTDTS S STVEWAIAELIRHPKILAQV
GAV84063.1 LKELDTVVGRDRLVTDLDLPQLTYLQAVIKETFRLHP
STPLSLPRVAAESCEIMGYHIPKGSTLLVNVWAIARDP
KEWAEPLEFRPERFLP GGEKPNVDIKGNDFEVIPF GAG
RRICAGMSLGLRMVQLLTATLVHAFDWDLTSGLMPE
DLSMEEAYGLTLQRAEPLMVHPRPRLSPNVY
Flavonoid 3' MA SFLLY S IL SAVFLYFIFATLRKRHRLPLPPGPKPWPII 50 hy droxyl as e (F3 'H) GNLPHMGPVPHHSLAALAKVYGPLMHLRLGFVDVV
VAA SA S VAAQFLKVHDANF SSRPPNSGAKYVAYNYQ
DLVFAPYGPRWRMLRKISSVHLF SGKALDDFRHVRQ
Theobroma cacao DEVGVLVRALADAKTKVNLGQLLNVCTVNALGRVM
LGKRVF GD GS GKADPEADEFK SMVVELMVLAGVVNI

GDFIPALEWLDLQGVQAKMKKLHKRFDRFLSAILEEH
KIKARDGSGQHKDLLSTFISLEDADGEGGKLTDTEIKA
Accession:
LLLNIVIFTAGTDTSSSTVEWAIAELIRHPKILAQVRKEL
E0Y22049.1 DSVVGRDRLVSDLDLPNLTYFQAVIKETFRLHPSTPLS
LPRMASESCEINGYHIPKGATLLVNVWAIARDPDEWK
DPLEFRPERFLPGGERPNADVRGNDFEVIPFGAGRRIC
AGMSLGLRMVQLLAATLVHAFDWELADGLMPEKLN
MEEAFGLTLQRAAPLMVHPRPRLSPRAY
Flavonoid 3' MTPLTLLIGTCVTGLFLYVLLNRCTRNPNRLPPGPTPW 51 hydroxylase (F3 'H) PVVGNLPHLGTIPHHSLAAMAKKYGPLMHLRLGFVD
VVVAASASVAAQFLKTHDANFADRPPNSGAKHIAYN
YQDLVFAPYGPRWRMLRKICSVHLFSTKALDDFRHV
Gerbera hybrida RQEEVAILARALVGAGKSPVKLGQLLNVCTTNALAR
VMLGRRVFDSGDAQADEFKDMVVELMVLAGEFNIG
DFIPVLDWLDLQGVTKKMKKLHAKFDSFLNTILEEHK
Accession:
TGAGDGVASGKVDLLSTLISLKDDADGEGGKLSDIEI
ABA64468.1 KALLLNLFTAGTDTSSSTIEWAIAELIRNPQLLNQARK
EMDTIVGQDRLVTESDLGQLTFLQAIIKETFRLHPSTPL
SLPRMALESCEVGGYYIPKGSTLLVNVWAISRDPKIW
ADPLEFQPTRFLPGGEKPNTDIKGNDFEVIPFGAGRRIC
VGMSLGLRMVQLLTATLIHAFDWELADGLNPKKLNIVI
EEAYGLTLQRAAPLVVHPRPRLAPHVYETTKV
Flavonoid 3' MAPLLLLFFTLLLSYLLYYYFFSKERTKGSRAPLPPGP 52 hydroxylase (F3 'H) RGWPVLGNLPQLGPKPHHTLHALSRAHGPLFRLRLGS
VDVVVAASAAVAAQFLRAHDANFSNRPPNSGAEHIA
YNYQDLVFAPYGPGWRARRKLLNVHLFSGKALEDLR
Phoenix dactylifera PVREGELALLVRALRDRAGANELVDLGRAANKCATN
ALARAMVGRRVFQEEEDEKAAEFENIVIVVELMRLAG
VFNVGDFVPGIGWLDLQGVVRRMKELHRRYDGFLDG
LIAAHRRAAEGGGGGGKDLLSVLLGLKDEDLDFDGE

Accession: GAKLTDTDIKALLLNLFTAGTDTTS STVEWAL SELVK
XP 008791304.2 HPDILRKAQLELD S VVGGDRLV SE SDLPNLPFMQ AIIK
ETFRLHP STPL SLPRMAAEECEVAGYCIPKGATLLVNV
WAIARDPAVWRDPLEFRPARFLPDGGCEGMDVKGND
FGIIPFGAGRRICAGMSLGIRMVQFMTATLAHAFHWD
LPEGQMPEKLDMEEAYGLTLQRATPLMVHPVPRLAP
TAYQ S
Cytochrome P450 MA SN SNLIRAIESAL GVSF GSELVSDTAIVVVT T SVAVI 53 IGLLFFLLKRS SDRSKESKPVVISKPLLVEEEEEEDEVE
reductase (CPR) AGS GK TK VTMF YGT Q T GT AEGF AK SL AKEIKARYEK
AIVKVVDLDDYAADDDQYEQKLKKETLVFFMLATYG
Camellia sinensis DGEPTDDAARFYKWFTEENERGAWLQQLTYGVF SLG
NRQYEEIFNKIGKVVDEQL SKQGAKRLIPVGLGDDDQ
CIEDDFAAWRETLWPELDQLLRDEDDANTVSTPYAA
Accession: AIPEYRVVIHDPLSGRGEAP SF SID SHLTICEIWSTSREG

SVSDTAIVVVTT SVAVIIGLLFFLLKRS SDRSKESKPVV
ISKPLLVEEEEDEVEAGS GKTKVTLFYGTQ T GTAEGF A
KSLAEEIKARYEKAIVKVVDLDDYAADDDQYEQKLK
KETLVFFMLATYGDGEPTDNAARFYKWFTEENERGA
WLQQLTYGVF SLGNRQYEEIFNKIGKVVDEQL SK Q GA
KRLIPVGLGDDDQCIEDDFAAWRETLWPELDQLLRDE
DDANTVS TP YTAAIPEYRVVIHDP TT T SYEDKNLNMA
NGNASYDIHHPCRVNVAVQRELHKPESDRSCIHLEFDI
S GT GIIYET GDHVGVYADNFDEVVEEAANLLGQPLEL
LF S VHADKDD GT SLGGSLPPPFPGPCTLRDALAHYAD
LLNPPRKAAL S AL AAHAVEP SEAERLKFL S SP Q GKED
YSQWVVASQRSLLEIMAEFP SAKPPLGVFFAAVAPRL

C STWMKNAVPLEKSHDC S SAPIFTRT SNFKLPTDP SIPI

IIVIVGPGTGLAPFRGFLQERLALKEDGVQLGHAMLFFG
CRNRRMDFIYEDELNNF VD Q GAV SELVVAF SREGPEK
EYVQHKLNAKAAQ VW GLI S Q GGYL YVC GDAK GMAR
DVHRMLHTIVEQQENVD SRKAEVIVKKLQMEGRYLR
DVW
Cytochrome P450 MA SN SNLIRAIESAL GVSF GSELVSDTAIVVVT T SVAVI 54 IGLLFFLLKRS SDRSKESKPVVISKPLLVEEEEEEDEVE
reductase (CPR) AGS GK TK VTMF YGT Q T GT AEGF AK SL AKEIKARYEK
AIVKVVDLDDYAADDDQYEQKLKKETLVFFMLATYG
Cephalotus DGEPTDDAARFYKWFTEENERGAWLQQLTYGVF SLG
folhcularis NRQYEHFNKIGKVVDEQL SKQGAKRLIPVGLGDDDQ
CIEDDFAAWRETLWPELDQLLRDEDDANTVSTPYAA
AIPEYRVVIHDPLSGRGEAP SF SID SHLTICEIWSTSREG
Accession: SNQQISEYFWT SNSLKTMASNSNLIRSIESALGVSFGSE
GAV59576.1 SVSDTAIVVVTT SVAVIIGLLFFLLKRS SDRSKESKPVV
I SKPLLVEEEEDEVEAGS GKTKVTLFYGTQ T GTAEGF A
KSLAEEIKARYEKAIVKVVDLDDYAADDDQYEQKLK
KETLVFFMLATYGDGEPTDNAARFYKWFTEENERGA
WLQQLTYGVF SLGNRQYEHFNKIGKVVDEQL SK Q GA
KRLIPVGLGDDDQCIEDDFAAWRETLWPELDQLLRDE
DDANTVS TP YTAAIPEYRVVIHDP TT T SYEDKNLNMA
NGNA SYDIREIP CRVNVAVQRELHKPE SDR S CIHLEFDI
S GT GIIYET GDHVGVYADNFDEVVEEAANLLGQPLEL
LF S VHADKDD GT SLGGSLPPPFPGPCTLRDALAHYAD
LLNPPRKAAL S AL AAHAVEP SEAERLKFL S SP Q GKED
YSQWVVASQRSLLEIMAEFP SAKPPLGVFFAAVAPRL
QPRYYSIS S SPRFVPNRVHVTCALVYGP SP TGRIHKGV
C STWMKNAVPLEKSHDC S SAPIFTRT SNFKLPTDP SIPI
IIVIVGPGTGLAPFRGFLQERLALKEDGVQLGHAMLFFG
CRNRRMDFIYEDELNNF VD Q GAV SELVVAF SREGPEK

EYVQHKLNAKAAQVWGLISQGGYLYVCGDAKGMAR
DVHRMLHTIVEQQENVDSRKAEVIVKKLQMEGRYLR
DVW
Cytochrome P450 MSSSSSSPFDLMSAIIKGEPVVVSDPANASAYESVAAE 55 LSSMLIENRQFAMIISTSIAVLIGCIVMLLWRRSGGSGS
reductase (CPR) SKRAETLKPLVLKPPREDEVDDGRKKVTIFFGTQTGT
AEGFAKALGEEARARYEKTRFKIVDLDDYAADDDEY
Brass/ca napus EEKLKKEDVAFFFLATYGDGEPTDNAARFYKWFTEG
DDRGEWLKNLKYGVFGLGNRQYEHFNKVAKVVDDI
LVEQGAQRLVHVGLGDDDQCIEDDFTAWREALWPEL
Accession: DTILREEGDTAVTPYTAAVLEYRVSIHNSADALNEKN
XP 013706600.1 LANGNGHAVFDAQHPYRANVAVRRELHTPESDRSCT
HLEFDIAGSGLTYETGDHVGVLSDNLNETVEEALRLL
DMSPDTYFSLHSDKEDGTPISSSLPPTFPPCSLRTALTR
YACLLSSPKKSALLALAAHASDPTEAERLKHLASPAG
KDEYSKWVVESQRSLLEVMAEFPSAKPPLGVFFAAV
APRLQPRFYSISSSPKIAETRIHVTCALVYEKMPTGRIH
KGVCSTWMKSAVPYEKSENCCSAPIFVRQSNFKLPSD
SKVPIIIVIIGPGTGLAPFRGFLQERLALVESGVELGPSVL
FFGCRNRRMDFIYEEELQRFLESGALSELSVAFSREGP
TKEYVQHKMMDKASDIWNMISQGAYVYVCGDAKG
MARDVHRSLHTIAQEQGSMDSTKAESFVKNLQMSGR
YLRDVW
Flavonoid 3', 5'- MALDTFLLRELAAAAVLFLISHYLIHSLLKKSTPPLPPG 56 hydroxylase PKGWPFVGALPLLGTMPHVALAQMAKKYGPVMYLK
(F3'5'H) MGTCGMVVASTPDAARAFLKTLDLNFSNRPPNAGAT
HLAYNAQDMVFADYGPRWKLLRKLSNLHMLGGKAL
EDWTQVRTVELGHMIQAMCEASRAKEPVVVPEMLTY
AMANIVIIGKVILGHRVFVTQGSESNEFKDMVVELMTS

Cephalotus AGYFNIGDFIPSIAWMDLQGIERGMKKLHKRFDALLT
folhcularis KMFEEHMATAHERKGNPDLLDIVMANRDNSEGERLT
TTNIKALLLNLF SAGTDTS S SIIEW SLAEMLKNP SILKR
AHEEMDQVIGRNRRLEESDIKKLPYLQAICKESFRKHP
Accession: STPLNLPRVS SQACQVNGYYIPKDTRL SVNIWAIGRDP

RICAGTRMGIVLVEYILGTLVHSFDWSLPHGVKLNMD
EAFGLALQKAVPLAAIVSPRLAPTAYVV
Flavonoi d 3 ' , 5'- MSIFLIT SLLLCL SLHLLLRRRHISRLPLPPGPPNLPIIGA 57 hydroxylase LPFIGPMPHSGLALLARRYGPIMFLKMGIRRVVVA S SS
(F3'5'H) TAARTFLKTFDSHFSDRPSGVISKEISYNGQNMVFADY
GPKWKLLRKVS SLHLLGSKAMSRWAGVRRDEAL SMI
QFLKKHSDSEKPVLLPNLLVCAMANVIGRIAMSKRVF
Dendrobium HEDGEEAKEFKEMIKELLVGQGASNMEDLVPAIGWL
moniliforme DPMGVRKKMLGLNRRFDRMVSKLLVEHAETAGERQ
GNPDLLDLVVASEVKGEDGEGLCEDNIKGFISDLFVA
GTDT SAIVIEWAMAEMLKNP SILRRAQEETDRVIGRH
Accession:
RLLDESDIPNLPYLQAICKEALRKHPPTPLSIPHYASEP

RFLQGEMARIDPMGNDFELIPFGAGRRICAGKLAGMV
MVQYYLGTLVHAFDWSLPEGVGELDMEEGPGLVLPK
AVPLAVMATPRLPAAAYGLL
D i hy drofl av on ol 4- MGSEAE T VC VT GA S GF IGS WL IIVIRLLERGYT VRAT VR 58 reductase (DFR) DPDNEKKVKHLVELPKAKTHLTLWKADLSDEGSFDE
AIHGCTGVFHVATPMDFESKDPENEVIKPTINGVLGIIVI
KACKKAKTVKRLVF T S S AGTVDVEEHKKPVYDEN SW
Acer palmatum SDLDFVQ S VKMT GWMYF V SKTLAEKAAWKF AEEN S I
DFISVIPPLVVGPFLMP SMPP SLITAL SPITRNEGHYAII
KQGNYVHLDDLCMGHIFLYEHAESKGRYF C S SH S ATI
LEL SKFLRERYPEYDLPTEYKGVDD SLENVVF C SKKIL

Accession: DLGFQFKYSLEDMFTGAVETCREKGLIPLTNIDKKHV
AWN08247.1 AAKGLIPNNSDEIHVAAAEKTTATA
Dihydroflavonol 4- MGSASETVCVTGAAGFIGSWLVMRLIQNGYKVRATV 59 reductase (DFR) RDPANMKKVKHLLELPNAKTNLSLWKADLAEEGSFD
EAIKGCTGVFHVATPMDFESKDPENEVIKPTINGLIDI
MKACMKAKTVRRLVFTSSAGTVDVTEHPKPLFDESC
Abrus precatorius WSDVQFCRRVRMTGWMYFVSKTLAEQEAWKFAKEN
NIDFISVIPPLVVGPFLVPTMPPSLITALSLITGNESHYAI
IKQGQFVEILDDLCLAHIFLFQHPKAQGRYICC SHEATI
Accession:
HDIASLLNQKYPEFNVPTKFKNIPDQLEIIRF S SKKITDL
XP 027329642.1 GFKFKYSLEDMFTGAVETCKEKRLLSETAEISGTTQK
Dihydroflavonol 4- MENEKKGPVVVTGASGYVGSWLVMKLLQKGYEVRA 60 reductase (DFR) TVRDPTNLKKVKPLLDLPRSNELLSIWKADLDGIEGSF
DEVIRGSIGVFHVATPMNFQ SKDPENEVIQPAINGLLGI
LRSCKNAGSVQRVIFTSSAGTVNVEEHQAAAYDETC
Dendrobium WSDLDFVNRVKMTGWMYFLSKTLAEKAAWEFVKD
moniliforme NHIHLITIIPTLVVGSFITSEMPPSMITALSLITGNDAHY
SILKQIQFVHLDDLCDAHIFLFEHPKANGRYICSSYDST
IYGLAEMLKNRYP TYAIPHKFKEIDPDIKCV SF S SKKL
Accession:
MELGFKYKYTMEEMFDDAIKTCREKKLIPLNTEEIVL
AEB96144.1 AAEKFEEVKEQIAVK
Dihydroflavonol 4- MASESESVCVTGASGFVGSWLVMRLLDRGYTVRATV 61 reductase (DFR) RDPANKKKVKHLLDLPKAATHLTLWKADLAEEGSFD
EAIKGCTGVFHVATPMDFESKDPENEVIKPTINGVLDI
MKACLKAKTVRRLVFTASAGSVNVEETQKPVYDESN
Rosa chinensis WSDVEFCRRVKMTGWMYFASKTLAEQEAWKFAKEN
NIDFITIIPTLVIGPFLMPAMPPSLITGLSPLTGNESHYSII

Accession: KQGQFIEILDDLCQSHIYLYEHPKAEGRYICSSHDATIH
XP 024167119.1 EIAKLLREKYPEYNVPTTFKGIEENLPKVHF S SKKLLE
TGFEFKYSLEDMFVGAVDACKAKGLLPPPTERVEKQE
VDES SVVGVKVTG
Leuco anthocy ani di MTV S SP C VGEGQ GRVLIIGA SGFIGEFIAQA SLD SGRTT 62 n reductase (LAR) FLLVRSLDKGAIPSKSKTINSLHDKGAILIHGVIEDQEF
VEGILKDHKIDIVISAVGGANILNQLTIVKAIKAVGTIK
RF LP SEF GHD VD RANP VEP GL AMYKEKRMVRRLIEE S
Camellia sinensis GVPYTYIC CN S IA SWPYYDNTHP SEVIPPLDRFQIYGD
GTVKAYF VD GSDIGKF TMKVVDDIRTLNK S VHF RP SC
NFLNMNEL S SLWEKKIGYMLPRLTVTEDDLLAAAAE
Accession:
NIIPQ SIVA SF THDIF IK GC Q VNF SID GPNEVEV SNL YPD
XP 028127206.1 ETFRTMDECFDDFVMKMDRWN
Leucoanthocyanidi MTRSPSPNGQAEKGSRILIIGATGFIGHFIAQASLASGK 63 n reductase (LAR) STYILSRAAARCPSKARAIKALEDQGAISIHGSVNDQE
FMEKTLKEHEIDIVISAVGGGNLLEQVILIRAMKAVGT
IKRFLP SEFGHDVDRAEPVEPGLTMYNEKRRVRRLIEE
Coffea arabica SGVPYTYICCNSIASWPYYDNTHP SEVSPPLDQFQIYG
D GS VKAYF VAGADIGKF T VKATED VRTLNKIVHF RP S
CNFLNINELATLWEKKIGRTLPRVVVSEDDLLAAAEE
Accession:
NIIPQ SVVA SF THDIFIKGCQVNFPVDGPNEIEVS SLYP
XP 027097479.1 DEPFQTMDECFNEFAGKIEEDKKHVVGTKGKNIAHRL
VDVLTAPKLCA
L eu c o anth o cy ani di MK S TNMNGS SPNVSEETGRTLVVGSGGFMGRFVTEA 64 n reductase (LAR) SLD S GRP TYILARS S SN SP SKASTIKFLQDRGATVIYGSI
TDKEFMEKVLKEHKIEVVISAVGGGSILDQFNLIEAIR
NVD TVKRF LP SEF GHD TDRADP VEP GL TMYE QKRQ IR
Theobroma cacao RQ IEK S GIP YTYIC CN SIAAWPYHDNTHP AD VLPPLDR
FKIYGDGTVKAYFVAGTDIGKF TIM S IEDDRTLNKTVH

Accession: FQPP SNLLNINEMASLWEEKIGRTLPRVTITEEDLLQM
ADD 5 1357.1 AKEMRIPQ SVVAALTHDIFINGCQINF SLDKPTDVEVC
SLYPDTPFRTINECFEDFAKKIIDNAKAVSKPAASNNAI
FVPTAKPGALPITAICT
L euco anthocy ani di MTV SP S IA SAAK S GRVLIIGATGF IGKF VAEA SLD S GLP 65 n reductase (LAR) TYVLVRPGP SRP SKSDTIKSLKDRGAIILHGVMSDKPL
MEKLLKEHEIEIVISAVGGATILDQITLVEAIT SVGTVK
RFLP SEFGHDVDRADPVEPGLTMYLEKRKVRRAIEKS
Fragaria x GVPYTYICCNSIASWPYYDNKHP SEVVPPLDQFQIYGD
ananassa GTVKAYF VD GPDIGKF TMK T VDDIRTMNKNVHFRP S S
NLYDINGLASLWEKKIGRTLPKVTITENDLLTMAAEN
RIPE SIVASF THD IF IKGCQTNFPIEGPNDVDIGTL YPEE
Accession:
SFRTLDECFNDFLVKVGGKLETDKLAAKNKAAVGVE
ABH07785.2 PMAITATC A
Anthocyanin MTQNKEPVNQGKSEHDEQRVESLAS SGIESIPKEYVRL 66 dioxygenase (ANS) NEELT SMGNVFEEEKKEEGSQVPTIDIKDIASEDPEVR
GKAIQELKRAAMEWGVMHLVNHGISDELIDRVKVAG
QTFFELPVEEKEKYANDQASGNVQGYGSKLANSASG
Chenopodium RLEWEDYYFHL SYPEDKRDL SIWPETP ADYIP AV SEY S
quinoa KELRYL ATKIL SAL SLALGLEEGRLEKEVGGLEELLLQ
FKINYYPKCPQPELALGVEAHTDVSALTFILHNMVPG
LQLFYEGKWVTAKCVPNSIIMHIGDTIEIL SNGKYK S IL
Accession:
HRGLVNKEKVRISWAVFCEPPKEKIILKPLPDLVSDEE
)CP 021735950.1 PARYPPRTF AQHVQYKLFRKT Q GP Q TTITKN

Anthocyanin MASSKVMPAPARVESLASSGLASIPTEYVRPEWERDD 67 dioxygenase (ANS) SLGDALEEIKKTEEGPQIPIVDLRGFDSGDEKERLHCM
EEVKEAAVEWGVMHIVNHGIAPELIERVRAAGKGFFD
LPVEAKERYANNQSEGKIQGYGSKLANNASGQLEWE
Iris sanguinea DYFFHLIFPSDKVDLSIWPKEPADYTEVMMEFAKQLR
VVVTKMLSILSLGLGFEEEKLEKKLGGMEELLMQMKI
NYYPKCPQPELALGVEAHTDVSSLSFILHNGVPGLQV
Accession:
FHGGRWVNARLVPGSLVVHVGDTLEILSNGRYKSVL
QCI56004.1 HRGLVNKEKVRISWAVFCEPPKEKIVLEPLAELVDKR
SPAKYPPRTFAQHIQHKLFKKAQEQLAGGVHIPEAIQN
Anthocyanin MATQVASIPRVEMLASAGIQAIPTEYVRPEAERNSIGD 68 dioxygenase (ANS) VFEEEKKLEGPQIPVVDLMGLEWENEEVFKKVEEDM
KKAASEWGVMHIINHGISMELMDRVRIAGKAFFDLPI
EEKEMYANDQASGKIAGYGSKLANNASGQLEWEDYF
Magnolia sprengeri FHLIFPEDKRDMSIWPKQPSDYVEATEEFAKQLRGLV
TKVLVLLSRGLGVEEDRLEKEFGGMEELLLQMKINYY
PKCPQPDLALGVEAHTDVSALTFILHNMVPGLQVFFD
Accession:
DKWVTAKCIPGALVVHIGDSLEILSNGKYRSILHRGLV
AHU88620.1 NKEKVRISWAIFCEPPKEKVVLQPLPELVSEAEPARFT
PRTFSQHVRQKLFKKQQDALENLKSE
Anthocyanin MVSSAAVVATRVERLATSGIKSIPKEYVRPQEELTNIG 69 dioxygenase (ANS) NVFEEEKKEGPEVPTIDLTEIESEDEVVRARCHETLKK
AAQEWGVMNLVNHGIPEELLNQLRKAGETFFSLPIEE
KEKYANDQASGKIQGYGSKLANNASGQLEWEDYFFH
Prosopis alba LVFPEDKCDLSIWPRTPSDYIEVTSEYARQLRGLATKI
LGALSLGLGLEKGRLEEEVGGMEELLLQMKINYYPIC
PQPELALGVEAHTDVSSLTFLLHNMVPGLQLFYNGQ
Accession:
WITAKCVPNSIFMHIGDTVEILSNGRYKSILHRGLVNK

XP 028787846.1 EKVRISWAVFCEPPKEKIILKPLPELVTDDEPARFPPRT
FAQHIQHKLFRKCQEGL SK
Anthocy ani di n-3 - MP QF TTNEPHVAVLAFPFGTHAAPLITIIHRLAVASPN 70 0-gly cotran sferase THF SFLNT SQ SNNSIF S SD VYNRQPNLKAHNVWD GVP
(3 GT) EGYVFVGKPQESIELFVKAAPETFRKGVEAAVAETGR
KVSCLVTDAFFWFAAEIAGELGVPWVPFWTAGPC SL S
THVYTDLIRK TIGVGGIEGREDE SLEFIP GM S Q VVIRDL
Cephalotus QEGIVFGNLESVF SDMVHRMGIVLPQ AAAIF IN SFEEL
folhcularis DLTITNDLKSKFKQFL SIGPLNLASPPPRVPDTNGCLP
WLD Q QKVA S VAYISF GT VMAP SPPELVALAEALEASK
IPFIW SLGEKLKVHLPKGFLDKTRTHGIVVPWAPQ SDV
Accession:
LENGAVGVFITHCGWNSLLESIAGGVPMICRPFFGDQ
GAV66155.1 RLNGRMVQDVWEIGVTATGGPFTTEGVMGDLDLIL S
QARGKKMKDNISVLKTLAQTAVGPEGS SAKNYEALL
NLVRL S I
Anthocy ani di n-3 - MAP QPIDDDHVVYEHHVAAL AFPF S THA SP TLALVRR 71 0-gly cotran sferase LAAASPNTLF SFF STSQ SNNSLF SNTITNLPRNIKVFDV
(3 GT) AD GVPD GYVF AGKP QEDIELFMKAAPHNF TT SLD T CV
AHTGKRLTCLITDAFLWFGAHLAHDLGVPWLPLWLS
GLNSL SLHVHTDLLRHTIGTQ SIAGRENELITKNVNIPG
Prunus cerasifera MSKVRIKDLPEGVIFGNLDSVFSRMLHQMGQLLPRAN
AVLVNSFEELDITVTNDLKSKFNKLLNVGPFNLAAAA
SPPLPEAPTAADDVTGCL SWLDKQKAAS S VVYV SF GS
Accession:
VARPPEKELLAMAQALEASGVPFLWSLKDSFKTPLLN
AKV89253.1 ELLIKASNGMVVPWAPQPRVLAHASVGAFVTHCGWN
SLLETIAGGVPMICRPFFGDQRVNARLVEDVLEIGVTV

ED GVF TKHGLIKYFDQVL SQQRGKKMRDNINTVKLL
AQ QPVEPK GS S AQNFKLLLDVI S GS TKV
Anthocy ani di n-3 - MVFQ SHIGVLAFPFGTHAAPLLTVVQRLATS SPHTLF S 72 0-glycotransferase FFN S AV SN S TLFNNGVLD SYDNIRVYHVWDGTPQGQ
(3 GT) AFTGSHFEAVGLFLKASPGNFDKVIDEAEVETGLKISC
LITDAFLWFGYDLAEKRGVPWLAFWTSAQCAL SAHM
YTHEILKAVGSNGVGETAEEELIQ SLIP GLEMAHL SDL
Scutellaria PPEIFFDKNPNPLAITINKMVLKLPKSTAVILNSFEEIDP
baicalensis ITT TDLK SKFHHFLNIGP S IL S SP TPPPPDDK TGCLAWLD
S Q TRPK S VVYI SF GT VITPPENEL AAL SEALET CNYPFL
WSLNDRAKKSLPTGFLDRTKELGMIVPWAPQPRVLA
Accession:
HRS VGVF VTHC GWN S ILE SIC SGVPLICRPFFGDQKLN

GEIIRENVNEMNEKAKIAVEPKGS SFKNFNKLLEIINAP
Q SS
Anthocy ani di n-3 - MS Q TT TNPHVAVLAFPF STHAAPLLAVVRRLAAAAPH 73 0-gly cotransferase AVF SFF STSQ SNASIFHD SMHTMQCNIKSYDISDGVPE
(3 GT) GYVFAGRP QEDIELF TRAAPE SFRQ GMVMAVAET GRP
V S CLVADAFIWF AADMAAEMGLAWLPFWTAGPN SL S
THVYIDEIREKIGV S GIQ GREDELLNF IP GM SKVRFRDL
Vitis vinifera QEGIVFGNLNSLF SRMLHRMGQVLPKATAVFINSFEEL
DD SLTNDLKSKLKTYLNIGPFNLITPPPVVPNTTGCLQ
WLKERKPT S VVYI SF GT VT TPPP AEVVAL SEALEASRV
Accession:
PFIWSLRDKARVHLPEGFLEKTRGYGMVVPWAPQAE

DQRLNGRMVEDVLEIGVRIEGGVFTKSGLMSCFDQIL

SQEKGKKLRENLRALRETADRAVGPKGS STENFITLV
DLVSKPKDV
Acetyl -C oA MPPPDHK AV S QFIGGNPLET AP A SPVADFIRKQ GGH S 74 carb oxyla se (ACC) VITKVLICNNGIAAVKEIRS IRKWAYETF GDERAIEF TV
MATPEDLKVNADYIRMADQYVEVPGGSNNNNYANV
DLIVDVAERAGVHAVWAGWGHASENPRLPESLAASK
Ustilago maydis HKIIFIGPPGSAMRSLGDKIS STIVAQHADVPC1VIPWSG

KIGYPVMIKASEGGGGKGIRKCTNGEEFKQLYNAVLG
EVPGSPVFVMKLAGQARHLEVQLLADQYGNAISIFGR
Accession:
DC SVQRRHQKIIEEAPVTIAPEDARESMEKAAVRLAK
XP 011390921.1 LVGYV S AGTVEWLY SPE S GEF AFLELNPRLQVEHP TT
EMVSGVNIPAAQLQVAMGIPLYSIRDIRTLYGMDPRG
NEVIDFDF S SPE SFKT QRKP QP Q GHVVACRITAENPD T
GFKPGMGALTELNFRS STSTWGYF SVGTSGALHEYAD
SQFGHIFAYGADRSEARKQMVISLKELSIRGDFRTTVE
YLIKLLETDAFESNKITTGWLDGLIQDRLTAERPPADL
AVICGAAVKAHLLARECEDEYKRILNRGQVPPRDTIK
TVF SIDFIYENVKYNFTATRS SVS GWVLYLNGGRTL V
QLRPLTDGGLLIGL S GK SHP VYWREEVGMTRLMID SK
TCLIEQENDPTQIRSP SPGKL VRFLVD S GDHVKANQ AI
AEIEVMKMYLPLVAAED GVV SF VK TAGVAL SP GDIIG
IL SLDDP SRVQHAKPFAGQLPDFGMPVIVGNKPHQRY
TALVEVLNDILDGYDQ SFRMQAVIKELIETLRNPELPY
GQASQIL S SLGGRIPARLEDVVRNTIEMGHSKNIEFPA
ARLRKLTENFLRDSVDPAIRGQVQITIAPLYQLFETYA
GGLKAHEGNVLASFLQKYYEVESQFTGEADVVLELR
LQADGDLDKVVALQT SRNGINRKNALLLTLLDKHIKG
TSLVSRTSGATMIEALRKLASLQGKSTAPIALKAREVS

LDADMP SLADR S AQMQAILRGS VT S SKYGGDDEYHA
PSLEVLREL SD SQYSVYDVLHSFFGHREHHVAFAALC
TYVVRAYRAYEIVNFDYAVEDFDVEERAVLTWQFQL
PR SA S SLKERERQV S I SDL SMMDNNRRARPIRELRTGA
MT S C ADVADIPELLPKVLKFFK S SAGAS GAPINVLNV
AVVDQTDFVDAEVRSQLALYTNAC SKEF SAARVRRV
TYLLCQPGLYPFFATFRPNEQGIWSEEKAIRNIEPALA
YQLELDRVSKNFELTPVPVS S S TIHLYF ARGIQN S AD T
RFFVRSLVRPGRVQ GDMAAYLI SE SDRIVND ILNVIEV
ALGQPEYRTADA SHIFM SF IYQLDV SLVDVQKAIAGFL
ERHGTRFFRLRITGAEIRMILNGPNGEPRPIRAFVTNET
GLVVRYETYEETVADD GS VILRGIEPQ GKDATLNAQ S
AHF'PYTTKVALQ SRRSRAHAL Q T TF VYDF ID VLGQAV
RA SWRKVAA SKIP GDVIK S AVELVFDEQENLREVKRA
PGMNNIGMVAWLVEVLTPEYPAGRKLVVIGNDVTIQ
AGSF GPVEDRFF AAA SKLAREL GVPRLYI SAN S GARIG
LATEALDLFKVKFVGDDPAKGFEYIYLDDESLQAVQA
KAPN SVMTKPVQAAD GS VHNIITDIIGKP Q GGL GVEC
L S GS GLIAGET SRAKD Q IF TATIITGR S VGIGAYLARLG
ERVIQVEGSPLILTGYQALNKLLGREVYTSNLQLGGPQ
IMYKNGVSHLTAQDDLDAVRSFVNWISYVPAQRGGP
LPIMPTTD SWDRAVTYQPPRGPYDPRWLINGTKAEDG
TKLTGLFDEGSFVETLGGWAT SVVTGRARLGGIPVGV
IAVETRTLERVVPADPANPNSTEQRIMEAGQVWYPNS
AYKTAQAIWDFDKEGLPLVILANWRGF SGGQQDMYD
EILKQGSKIVDGL S SYKQPVFVHIPPMGELRGGSWVV
VD SAINDNGMIEMSADVNSARGGVLEASGLVEIKYRA
DKQRATMERLD SVYAKLSKEAAEATDF TAQTTARKA
LAEREKQLAPIFTAIATEYADAHDRAGRMLATGVLRS
ALPWENARRYFYWRLRRRLTEVAAERTVGEANPTLK

HVERLAVLRQF VGAAA SDDDKAVAEHLEA SAD QLLA
A SKQLKAQYILAQI S TLDPELRAQLAA SLK
Acetyl-CoA MVDHK SLP GHF L GGN S VD TAP QDP VCEF VK SHQGHT 75 carb oxylase (ACC) VI SKVL IANNGMAAMKEIR S VRKWAYETF GNERAIEF
TVMATPEDLKANAEYIRMADNYIEVPGGTNNNNYAN
VELIVDVAERTGVHAVWAGWGHASENPRLPEMLAK S
Hesseltinella KNKCVFIGPPASAMRSLGDKIS STIVAQ SAD VP TMGW
vesiculosa S GD GV SET TTDHNGHVLVNDDVYN S AC VKTAEAGLA
SAEKIGFPVMIKASEGGGGKGIRKVEDP STFKQAFAQ
VQGEIPGSPIFIMKLAGNARHLEVQLLADQYGNAISLF
Accession:
GRDC SVQRRHQKIIEEAPVTIAKPDIFEQMEKAAVRLG
ORX57605 . 1 KLVGYVSAGTVEYLYSHHDEKFYFLELNPRLQVEHPT
TEMVSGVNLPAAQLQIAMGIPMHRIRDIRVLYGVQPN
SA SEIDF DLEHP TAL Q SQRRPMPKGHVIAVRITAENPD
AGFKP S GGVM QELNF R S STNVWGYF SVVS SGAMHEY
AD SQFGHIFAYGENRQQARKNIVIVIALKEL SIRGDFRT
TVEYIIRLLETPDF TDNTINT GWLDML I SKKL T AERPD T
MLAVF C GAVTKAHLA S VECW Q Q YKN SLERGQ IP SKE
SLKTVF TVDFIYENIRYNF T VTR S AP GIYTL YLNGTK T
QVGVRDL SD GGLL I SLNGR SHT T YNREEVQ ATRLMID
GK T CLLEKE SDP T QLR SP SP GKL V SLLLENGDHIRT GQ
AYAEIEVMKMYMPLVASEDGHVQFIKQVGATLEAGD
IIGIL SLDDP SRVKHALPF TGQVPKYGLPHLTGDKPHQ
RF THLKQTLEYVLQ GYDNQ GL IQ T IVKEL SEVLNNPEL
PYSEL S A SM SVL S GRIP GRLEQ QLHDLINQ AHAQNK G
FPAVDIQQAIDTFARDHLTTQAEVNAYKTAVAPIIVITIA
A SY SNGLKQHEH S VYVDLMEQYYNVEVLFN SNQ SRD
EEVILALRDQHKDDLEKVINIIL SHAKVNIKNNLILMLL
DIIYP AT S SEALDRC F LP ILKHL SEID SRGTQKVTLKAR
EYL IL C QLP SLEERQ SQMYNILK S SVTESVYGGGTEYR

TPSYDAFKDLIDTKFNVFDVLPNFFYHPDSYVSLAALE
VYCRRSYHAYKILDVAYNLEHQPYIVAWKFLLQ S SA
GGGFNNQRIASYSDLTFLLNKTEEEPIRTGAMVALKTL
EELEAELPRIMTAFEEEPLPPMLMKQPPPDKTEERMEN
ILNISIQGQDMEDDTLRKNMTTLIQAHSDAFRKAALR
RITLVVCRDNQTPDYYTFRERNGYEEDETIRHIEPALA
YQLELARLSNFDIKPCFIENRQMHVYYAVAKENPSDC
RFFIRALVRPGRVKSSMRTADYLISESDRLLTDILDTLE
IVSHDYKNSDCNHLFINFIPTFAIEADEVETALKDFVDR
HGKRLWKLRVTGAEIRFNIQSKRPDAPVIPLRFTVDNV
SGYILKVDVYQEVKTDKNGWILKSVGKIPGAMHMQP
LSTPYPTKEWLQPRRYKAHLMGTTYVYDFPELFRQAI
HNLWAQACKADAAVKIPSQVIEAKELVLDDDNQLQA
IDRAPGTNTVGMVAWLLTLRTPDYPRGRRVIAIANDI
TFKIGSFGVQEDLVFYKASEYARELGVPRVYLSANSG
ARIGLADELISRFHVAWKDEDQPGSGFEYLYLLPEEY
DALIQQGDAQSVLVQEVQDKGERRFRITDIIGHTDGL
GVENLRGSGLIAGATSRAYDDIF'TITLVTCRSVGIGAY
LVRLGQRTVQNEGQPIILTGAPALNKVLGREVYTSNL
QLGGTQIMYKNGVSHLTAENDLEGINKIMQWLSFVPE
CRGAPLPMRAGADPIDREIEYLPPKGPSDPRFFLAGKQ
ENGKWLSGFFDHGSFVETLSGWARTVVVGRARLGGI
PMGVVAVETRTVENIVPADPANADSQEQVVMEAGGV
WFPNSAYKTAQAINDFNKGEQLPLMIFANWRGFSGG
QRDMYNEVLKYGAQIVDALSNYKQPVFVYVVPNGEL
RGGAWVVVDSTINEDMMEMYADTQARGGVLEPEGI
VEIKYRRPQLLATMERLDPVYSDLKRRLAALDDSQKE
QADELIAQVEAREQALLPVYQQVAIQFADLHDRSGR
MEAKGVIRKTLEWRTARHYFYWRVRRRLLEEYAIRK
MDESRDQAKTLLQQWFQADTNLDDFDKNDQAVVA

WFDAKNLLLDQRIAKLKSEKLKDHVVQLASVDQDAV
VEGF SKLMESL S VD QRKEVLHKL ATRF
Acetyl -C oA MASTTPHD SRVVSVS SGKKLYIEVDDGAGKDAPAIVF 76 carboxylase (ACC) MHGLGS ST SFWEAPF SR SNL S SRFRLIRYDFDGHGL SP
V SLLDAADD GAMIPLVDLVEDLAAVMEWTGVDKVA
GIVGH SM S GL VA S TF AAKYPQKVEKL VLL GAMRSLN
Rhodotorula PTVQTNMLKRADTVLESGLSAIVAQVVSAAL SDK SK Q
toruloides D SPLAPAMVRTLVLGTDPLGYAAACRALAGAKDPDY

MD GVGHWHAVEDP AGLAKILD GFFL Q GKF SGEAKA
VNGSHAVDETPKKPKYDHGRVVKYLGGNSLESAPP S
Accession:
NVADWVRERGGHTVITKILIANNGIAAVKEIRSVRKW
GEM08739.1 AYETFGSERAIEFTVMATPEDLKVNADYIRMADQYVE
VPGGTNNNNYANVDVIVDVAERAGVHAVWAGWGH
A SENPRLPESLAASKHKIVF IGPPGSAMRSLGDKIS STI
VAQHAEVPCMDW S GQ GVD Q VT Q SLEGYVTVADDVY
QQACVHDADEGLARASRIGYPVMIKASEGGGGKGIR
KVEREQDFKQAFQAVLTEVPGSPVFIMKLAGAARHLE
VQVLADQYGNAISLFGRDC SVQRRHQKIIEEAPVTIAK
PDTFEQMEKSAVRLAKLVGYVSAGTVEFLYSAADDK
FAFLELNPRLQVEHPTTEMVSGVNLPAAQLQVAMGV
PLHRIRDIRTLYGKAPNGS SEIDFEFENPESAKTQRKP S
PKGHVVAVRITAENPDAGFKP SMGTLQELNFRS STNV
WGYF SVGSAGGLHEFAD SQFGHIFAYGSDRSESRKN
MVVALKEL S IRGDFRT TVEYLIKLLETDAFEQNTIT TA
WLD SLISARLTAERPDTTLAIICGAVTKAHLASEANIA
EYKRILEKGQ SPPKELLATVVPLEF VLEDVKYRATA SR
S SP S SW SIYVNGSNVSVGIRPL AD GGLLILLD GRSYT C
YAKEEVGALRL SID SRTVLVAQENDPTQLRSP SPGKL

FIKQPGATLEAGDILGILSLDDP SRVHHAKPFDGQLPA
LGLPSIIGTKPHQRFAYLKDVL SNILMGYDNQAIMQ S S
IKELISVLRNPELPYGEANAVL STL SGRIPAKLEQTLRQ
YID SAHESGAEFP SAKCRKAIDTTLEQLRPAEAQTVRN
FLVAFDDIVYRYRSGLKHHEWSTLAGIFAAYAETEKP
F SGKD SDVVLELRDAHRD SLD SVVKIVL SHYKAASKN
SLVLALLDVVKD SD SVPLIEQVVSPALKDLADLD SKA
TTKVALKAREVLIHIQLPSLDERLGQLEQILKASVTPT
VYGEPGHDRTPRGEVLKDVID SRFTVFDVLPSFFQHQ
DQWVSLAALDTYVRRAYRSYNLLNIEHIEADAAEDEP
ATVAWSFRMRKAASESEPPTPTTGLTSQRTASYSDLT
FLLNNAQ SEPIRYGAMF SVRSLDGFRQELGTVLRHF'P
D SNKGKLQQQPAAS SSQEQWNVINVALTVPASAQVD
EDALRADFAAHVNAMSAEIDARGMRRLTLLICREGQ
YPSYYTVRKQDGTWKELETIRDIEPALAFQLELGRLSN
FHLEPCPVENRQVHIYYATAKGNS SD CRFF VRALVRP
GRLRGNMKTADYLVSEADRLVTDVLD SLEVAS SQRR
AADGNHISLNFLYSLRLDFDEVQAALAGFIDRHGKRF
WRLRVTGAEIRIVLEDAQGNIQPIRAIIENVSGFVVKYE
AYREVTTDKGQVILKSIGPQGALHLQPVNFPYPTKEW
LQPKRYKAHVVGTTYVYDFPDLFRQAIRKQWKAVGK
TAPAELLVAKELVLDEFGKPQEVARPPGTNNIGMVG
WIYTIF TPEYP SGRRVVVIANDITFKIGSFGPEEDRYFY
AVTQLARQL GLPRVYL S AN S GARLGIAEELVDLF S VA
WADS SRPEKGFKYLYLTAEKLGELKNKGEKSVITKRI
EDEGETRYQITDIIGLQEGLGVESLKGSGLIAGETSRAY
DDIF'TITLVTARSVGIGAYLVRLGQRAVQVEGQPIILTG
AGALNKVLGREVYS SNLQLGGTQIMYKNGVSHLTAA
NDLEGVL SIVQWLAFVPEHRGAPLPVLP SPVDPWDR SI
DYTPIKGAYDPRWFLAGKTDEADGRWLSGFFDKGSF
QETLSGWAQTVVVGRARLGGIPMGAIAVETRTIERIIP

ADPANPL SNEQKIMEAGQVWYPNS SFKTGQAIFDFNR
EGLPLIIFANWRGF SGGQQDMFDEVLKRGSLIVDGL S
AYKQPVFVYIVPNGELRGGAWVVLDP SINAEGMMEM
YVDETARAGVLEPEGIVEIKLRKDKLLALMDRLDPTY
HALRVK STDASL SPTDAAQAKTELAAREKQLMPIYQQ
VALQF AD SHDKAGRIL SKGCAREALEW SNARRYFYA
RLRRRLAEEAAVKRLGEADPTLSRDERLAIVHDAVGQ
GVDLNNDLAAAAAFEQGAAAITERVKLARATT VAST
LAQLAQDDKEAFAASLQQVLGDKLTAADLARILA
Malonyl-CoA MNANLFSRLFDGLVEADKLAIETLEGERISYGDLVAR 77 synthase (matB) SGRMANVLVARGVKPGDRVAAQAEKSVAALVLYLA
TVRAGAVYLPLNTAYTLHELDYFIGDAEPKLVVCDPA
KREGIAALAQKVGAGVETLDAKGQGSLSEAAAQASV
Rhodopseudomonas DFATVPREGDDLAAILYT S GT T GRSKGAML SHDNLA S
pa/list/is NSLTLVEFWRF TPDDVLIHALPIYHTHGLF VA SNVTLF
Accession: ARASMIFLPKFDPDAIIQLMSRASVLMGVPTFYTRLLQ
WP 011661926.1 SD GL TKEAARHMRLF I S G SAPLLAD THREWA SRTGHA
VLERYGMTETNMNTSNPYDGARVPGAVGPALPGVSL
RVVDPET GAEL SP GEIGMIEVKGPNVF QGYWRMPEKT
KAEFRDDGFFITGDLGKIDADGYVFIVGRGKDLVITGG
FNVYPKEVESEIDAISGVVESAVIGVPHADLGEGVTAV
VVRDKGA S VDEAAVLGAL Q GQLAKFKMPKRVLF VD
DLPRNTMGKVQKNVLREAYAKLYAK
Malonyl-CoA MVNHLFDAIRL SIT SPE S TFIELED GKVWTYGAMFNC S 78 synthase (matB) ARITHVLVKLGVSPGDRVAVQVEKSAQALMLYLGCL
RAGAVYLPLNTAYTPAELEYFLGDATPKLVVVSPCAA
EQLEPLARRVGTRLLTLGVNGDGSLMDMASLEPVEF
Rhizobium ADIERKADDLAAILYT S GT TGR SKGAML THDNLL SNA
sp. BUS003 QTLREHWRF T SADRLIHALPIFHTHGLFVATNVTLLAG
GAIYLL SKFDPDQIFALMTRATVMMGVPTFYTRLLQD

ERLNKANTRHMRLF I S GSAPLLAETHRLFEEYTGHAIL
ERYGMTETNMIT SNP CD GARVP GTVGYALPGV S VRIT
Accession:
DPVSGEPLAAGEPGMIEVKGPNVFQGYWNMPDKTKE
NKF42351. 1 EFRSDGYFTTGDIGVMETDGRISIVGRGKDLIISGGYNI
YPKEIENEIDAIEGVVESAVIGVPHPDLGEGVTAIVVG
QPKAHLDL TTITNNL Q GRLARFKQPKNVIF VDELPRNT
MGKVQKNVLRDRYRDLYLK
Mal onyl-C oA MANHLFDLVRANATDLTKTFIETETGLKLTYDDLMT 79 synthase (matB) GTARYANVLVGLGVKPGDRVAVQVEKSAGAIFLYLA
CVRAGAVFLPLNTAYTLTEIEYFLGDAEPALVVCDPA
RRDGITEVAKKTGVPAVETLGKGQDGSLFDKAAAAP
Ochrobactrum sp. ETFADVARGP GDLAAILYT S GT TGRSKGAML SHDNLA

LVAGASMLFLPKFDADKVFELMPRATTMMGVPTFYV
RLVQDARL TREATKHMIRLF I S GS APLLAETHKLFREK
Accession:
TGVSILERYGMTETNMNT SNPYDGDRVAGTVGFPLPG
WP 114216069.1 VALRVADPETGAAIPQGEIGVIEVKGPNVF SGYWRMP
EKTAAEFRQD GEE ITGDLGKIDD Q GYVHIVGRGKDLV
IS GGYNVYPKEVETEID GMAGVVE S AVIGVPHPDF GE
GVTAVVVAEKGASLDEATIIKTLEQRLARYKLPKRVI
VVDDLPRNTMGKVQKNLLRDAYKGLYGG
Mal onate M SPELIS ILVLVVVF VIATTRS VNMGALAF AAAF GVGT 80 transporter (matC) LVADLDAD GIFAGFP GDLF VVLVGVTYLF AIARANGT
TDWLVHAAVRLVRGRVALIPWVMF AL T GALTAIGAV
SPAAVAIVAPVAL SF ATRY SI SPLLMGTMVVHGAQAG
Rhizobiales GF SPISIYGSIVNGIVEREKLPGSEIGLFLASLVANLLIA
bacterium AVLF AVL GGRKLWARGAVTPEGD GAP GKAGT GTT GS
GSD T GT GTGT GTGT S AGT GGTAP TAVAVRSDRET GG
AEGTGVRLTPARVATLVALVALVVAVLGFDLDAGLT
Accession:
AVTLAVVL STAWPDD SRRAVGEIAWSTVLLICGVLTY

MBN8942514.1 VGVLEEMGTITWAGEGVGGIGVPLLAAVLLCYIGAIV
SAFAS SVGIIVIGALIPLAVPFLAQGEIGAVGMVAALAV
SATVVDVSPF STNGALVLAAAPDVDRDRFFRQLMVY
GGIVVAAVPALAWLVLVVPGFG
Mal onate MGIELLSIGLLIAMFIIATIQPINMGALAFAGAFVLGSMI 81 transporter (matC) IGMKTNEIFAGFPSDLFLTLVAVTYLFAIAQINGTIDWL
VECAVRLVRGRIGLIPWVMFLVAAIITGF GAL GPAAV
AILAPVAL SF AVQYRIHPVMMGLMVIHGAQAGGF SPI
Rhizobium SIYGGITNQIVAKAGLPF AP T SLFL S SFFFNLAIAVLVFF
leguminosarum VF GGARVMKHDPA SL GPLPELHPEGV S A SIRGHGGTP
AKPIREHAYGTAADTATTLRLNNERITTLIGLTALGIG
ALVFKFNVGLVAMTVAVVLALL SPKT QKAAIDKV SW
Accession:
STVLLIAGIITYVGVMEKAGTVDYVANGIS SLGMPLLV
AAC83457.1 ALLLCFTGAIVSAFAS S TALL GAIIPLAVPFLLQGHIS AI
GVVAAIAISTTIVDTSPF STNGALVVANAPDDSREQVL
RQLLIYSALIAIIGPIVAWLVFVVPGLV
Mal onate MNIEIL SIGLLVAIFIIATIQPINMGVLAF GC TF VLGSLII 82 transporter (matC) GMKPADIFAGFPADLFLTLVAVTYLFAIAQINGTIDWL
VERS VRMVRGRVGWIPWVMFLVAAIIT GF GAL GPAA
VAILAPVAL SF AVQYRIHPVLMGLMVIHGAQAGGF SPI
Agrobacterium vitis SIYGGITNQIVAKAGLPFAPT SLFLS SFFFNLAIAVLIFFI
FGGL SILKQRS S VKGPLPELHPEGIS A SIKGHGGTPAKP
FREHAYGTAADTQ SKVRLTTEKVTTLIGLTALGVGAL
Accession:
VFKFNVGLVAITVAVLLALL SP TT QKAAIDKV SW S TV
WP 180575084.1 LLISGIITYVGVMEKAGTIDYVAHGISSLGMPLLVALL
LCFTGAIVSAFAS STALLGAIIPLAVPFLLQGHISAVGV
VAAIAISTTIVDTSPF STNGALVVANAPDDQRDKVMR
QMLIYSALIALIGPVIAWLVFVVPGII

Mal onate MSIEIL SILLL VAMF VIATIQPINMGALAF AC TF VLGSLI 83 transporter (matC) IGMKTSDIFAGFP SDLFLTLVAVTYLFAIAQINGTIDWL
VECAVRMVRGHVAWIPWVMF VVAAIIT GF GAL GPAA
VAIL APVAL SFAVQYRIHPVMMGLMVIHGAQ AGGF SP
Neorhizobium sp. IS VYGGITNQ IVAKAGLPF AP T SLFL S SFFFNLAIAVLVF
FVFGGARIMKQAAGPTGPLPELHPEGVSAAIRGHGGT
PAKPIREHAYGTAAD TLQ TLRLTPEKVF TLIGLTAL GI
Accession:
GALVFKFNVGLVAITVAVALALISPKTQKAAVDKVS
WP 105370917.1 W S TVLLIAGIITYVGVLEKAGTVNYVANGIS SLG1VIPLL
VALLLCFTGAIVSAFAS STALL GAIIPLAVPFLLQGHIS
AVGVVAAIAISTTIVDT SPF STNGALVVANAPDETREQ
VLRQLLIY S ALIAIIGPVVAWL VF VVP GL V
Mal onate CoA- MT TWNQKQ QRKAQKL AKACD SGFDKYVPHERIIALL 84 transferase (MdcA) ETVIDRGDRVCLEGNNQKQADFL SK SL S SCNPDIVNG
LHIVQ S VL ALP SHIDVFERGIASKVDF SF AGP Q SLRLAQ
LVQAQKITIGAIHTYLELYGRYFIDLTPNVALITAHAA
Moraxella DKRGNLYTGANTEDTPAIVEATTFKSGIVIAQVNEIVD
catarrhahs ELPRVDIP SDWVDYYTQ SPKHNYIEPLFTRDPAQITEIQ
ILMAMMAIKGIYAPYKINRLNHGIGFD TAAIELLLP TY
AESLGLKGEIC THW ALNPHP TLIP AIES GF IHSVHSF GS
Accession:
EVGMENYVKARSDVFFTGADGSMRSNRAF SQTAGLY
WP 064617969.1 ACDLFIGSTLQIDLQGNSSTATADRIAGFGGAPNMGSD
PHGRRHASYAYMKAGREAVDGSPIKGRKLVVQMVE
TYREHMQ SVFVNELDAFKLQQKMGADLPPEVIIYGDD
VTHIVTEEGIANLLLCRTPDEREQAIRGVAGYTPIGLG
RDDTMVARLRERKVIQRPEDLGINPMHATRDLLAAKS
VKDLVRWSDRLYEPP SRFRNW

Mal onate CoA- MNAPQPRQWD SLRQNRARRLERAASLGLAGQNGKEI 85 transferase (MdcA) PVDRIIDLLEAVIQP GDRVCLEGNNQKQADFL SE SLAB
CDPARINHLSMVQ SVLALPSHVDLFERGLATRLDF SF S
GPQGARLAKLVQEQRIEIGAIHTYLELFGRYFMDLTPN
Dechloromonas VALIAAQAADAEGNLYLGPNTEDTPAIVEATAFKGGI
aromatica VIAQVNERLDKLPRVDVPADWVDFTVLAPKPNYIEPL
FTRDPAQITEVQVLMAMMAIKGIYAEYGVTRLNHGIG
FDTAAIELLLPTYAADLGLKGKICTHWALNPHPTLIPA
Accession:
IEAGFVESVHCFGSEVGMDDYISARSDIFFTGADGSMR
WP 011289741.1 SNRAF SQTAGLYACDMFIGSTLQMDLAGNS STATLGR
IT GF GGAPNMGSDPHGRRHA SPAWLKAGREAYGP QA
IRGRKLVVQMVETFREHMAPVFVDDLDAWKLQASM
GSDLPPIIVIIYGDD V SHIVTEEGIANLLL CRTPAEREQAI
RGVAGFTPVGMARDKGTVENLRDRGIIRRPEDLGIDP
RQASRDLLAARSIKDLVRC SGGLYAPP SRFRNW
Mal onate CoA- MSRQWDTQAD SRRQRLQRAAALAPQGRVVAADDVV 86 transferase (MdcA) ALLEAVIEPGDRVCLEGNNQKQADFLARCLTEVDPAR
VHDLHMVQ SVLSLAAHLDVFERGIAKRLDF SF S GP QA
ARLAGLVSEGRIEIGAIHTYLELFGRYFIDLTPRIALVT
Pseudomonas AQAADRHGNLYTGPNTEDTPVIVEATAFKGGIVIAQV
cissicola NEILDTLPRVDIPADWVDFVTQAPKPNYIEPLFTRDPA
QISEIQVLMAMMAIKGIYAEYGVDRLNHGIGFDTAAIE
LLLPTYAQ SLGLKGKICRHWALNPHPALIPAIESGFVQ
Accession:
SVH SF GSELGMENYIAARPDIFF TGAD GSMRSNRAL S
WP 078590875.1 QTAGLYACDMFIGSTLQIDLQGNS STATRDRIAGFGG
APNIVIGSDARGRRHASAAWLKAGREAATPGEMPRGR
KLVVQMVETFREHMAPAFVDRLDAWELAERANMPL
PPVMIYGDDVSHVLTEEGIANLLLCRTPEEREQAIRGV
SGYTAVGLGRDKRMVENLRDRGVIKRPDDLGIRPRD
ATRDLLAARTVKDLVRWSGGLYDPPKRFRNW

Mal onate CoA- MNKIYREKRSWRTRRDRKAKRIEHMKQIAKGKIIPTE 87 transferase (MdcA) KIVEALTALIFPGDRVVIEGNNQKQASFL SKALSQVNP
EKVNGLHIIIVISSVSRPEHLDLFEKGIARKIDF SYAGPQ S
LRMSQMLEDGKLVIGEIHTYLELYGRLFIDLTP S VAL V
Geobacillus AADKADASGNLYTGPNTEETPTLVEATAFRDGIVIAQ
subterraneus VNELADELPRVDIPGSWIDFVVAADHPYELEPLFTRDP
RLITEIQILMAMMVIKGIYERHNIQ SLNHGIGFNTAAIE
LLLPTYGESLGLKGKICKHWALNPHPTLIPAIETGWVE
Accession:
SIHCFGGEVGMEKYIAARPDIFFTGKDGNLRSNRTLSQ
WP 184319829.1 VAGQYAVDLFIGSTLQIDRDGNSSTVTNGRLAGFGGA
PNIVIGHDPRGRRHS SPAWLDMITSDHPAAKGRKLVVQ
MVETFQKGNRPVFVESLDAIEVGRSARLATTPIMIYGE
DVTHIVTEEGIAYLYKAS SLEERRQAIAAIAGVTPIGLE
RDPRKTEQLRRD GVVAFPEDL GIRRTDAKRSLLAAK S I
EELVEWSEGLYEPPARFRSW
Pantothenate kinase MLLTIDVGNTHTVLGLFDGEEIVEHWRISTDSRRTADE 88 (C oaX) LAVLLQGLMGTHPLLGMELGEGIDGIAIC STVPAVLH
ELREVSRRYYGDVPAILVEPGVKTGVPILMDNPKEVG
Streptomyces sp.

YTGGVIAP GIEI S VEAL GLRGAQLRKIEL ARPRS VIGK S
Accession: TVEAMQ SGILYGFAGQVDGVVQRMACELAPDPADVT
WP 095682415.1 VIATGGLAPMVLGEAAVIDHHEPWLTLIGLRLVYERN
AGRR
Pantothenate ki na se MTKLWLDLGNTRLKYWLTDD SGQVLDHAAEQHLQA 89 (CoaX) PAELLKGLTFRLERLNPDFIGVS SVLGQAVNNHVAESL
ERLQKPFEFAQVHAKHALMS SDYNPAQLGVDRWLQ
Streptomyces MLGIIEP SKKQCVIGCGTAVTIDLVDQGHHLGGYIFP SI
cinereus YLQRESLF S GTRQI S IID GTFD S ID S GTNT QDAVHHGIM
Accession: L S IVGAINETIHRYP QFEITMT GGDAHTFEPHL S A S VET
WP 188874884.1 RQDLVLAGLQRFFAAKNNTKNQN

Pantothenate kinase MLL TID VGNTQT TL GLFDGEEVVDHWRIS TDPRRT AD 90 (CoaX) ELAVLMQGLMGRQPGGAGRERVDGLAIC S SVPAVLH
ELREVTRRYYGDLPAVLVAPGVKTGVHVLMDNPKEV
Kitasatospora GADRIVNALAANHLYGGPCIVVDFGTATTFDAINERG
kifunensis DYVGGAIAPGIEISVEALGVRGAQLRKIELAKPRNVIG
Accession: KNTVEGMQ S GVLYGF AGQ VD GLVTRMAKEL SP TDPE
WP 184936930.1 DVQVIATGGLAPLVLDEAS SIDVHEPWLTLIGLRLVYE
RNTAS
glutamyl -tRNA MTLLALGINHKTAPVSLRERVTF SPDTLDQALDSLQA 91 reducta se (hemA) LPMVQGGVVLSTCNRTEIYL SVEEQDNLREALIRWLC
EYHNLNEEDLRNSLYWHQDNDAVSHLMRVASGLD S
LVL GEP QIL GQ VKKAF AD S QK GHQNA SALERMF QK S
Citrobacter F S VAKRVRTETDIG S S AV S VAF AAC TLARQIFE SL S TV
freundii TVLLVGAGETIELVARHLREHKVKKMIIANRTRERAQ
VLADEVGAEVISL SDIDARLQDADIIISSTASPLPIIGKG
MVERALKNRRNQPMLLVDIAVPRDVEPEVGKLSNAY
Accession:
LYSVDDLQ S II SHNL AQRKAAAVEAETIVEQEA SEFMA
NTY05430.1 WLRAQGASDTIREYRSQ SEQIRDELTAKALAALQQGG
DAQ AIIVIQDL AWKL TNRLIHAP TK SL Q Q AARD GD SER
LNILRDSLGLE
glutamyl -tRNA MTLLALGINHKTAPVSLRERVTF SPETIEQALS SLLQQP 92 reductase (hemA) LVQGGVVL STCNRTELYLSVEQQENLQEQLVKWLCD
YHHLSADEVRKSLYWHQDNAAVSHLMRVASGLDSL
VVGEP QIL GQ VKKAF AE S QHGQ AV S GELERLF QK SF S
Pseudomonas VAKRVRTETDIGA S AV S VAF AAC TL ARQIFE SL SD V S V
reactans LLVGAGETIELVARHLREHKVREIMMIANRTRERAQV
LASEVGAEVITLQDIDARLADADIIIS S TASPLPIIGK GM
VERALKARRNQPMLMVDIAVPRDIEPEVGKLANAYL
Accession:
YSVDDLHSIIQNNMAQRKAAAVQAESIVEQES SNFMA
NWA43040.1 WLR S Q GAVEIIRDYRSRADLVRAEAEAKALAAIAQ GA

D V S AVIHELAHKLTNRLIHAPTR SL Q QAA SD GDVERL
QILRDSLGLDQQ
glutamyl -tRNA MTLLAL GINHKTAPVALREKV SF SPDTMGDALNNLLQ 93 reducta se (hemA) QPAVRGGVVL STCNRTELYLSMEDKENSHEQLIRWLC
QYHQIEPNELQ S SIYWHQDNQ AV SHLMRVA S GLD SL
Gammaproteobacte VLGEPQIL GQVKKAF AD S QNYD SL S SELERLF QK SF SV
ria AKRVRTETQIGANAVSVAFAACTLARQIFESL S SL TILL
Accession: VGAGETIELVARHLREHQVKKIIIANRTKERAQRLA SE
WP 193016510.1 VDAEVITLSEIDECLAQADIVISSTASPLPIIGKGMVER
ALKKRRNQPMLLVDIAVPRDIEQDVEKLNNVYLY S V
DDLEAIIQHNREQRQAAAVQAEHIVQQESGQFMDWL
RAQGAVGAIREYRDSAETLRAEMTEKAITLIQNGADA
EKVIQQLSHQLMNRLIHTPTKSLQQAASDGDIERLNLL
RE SL GITHN
-ami nol evul i ni c MGPALDVRGKQLAAGYASVAGQADVEKIHQDQGITI 94 acid synthase PPNATVEMCPHAKAARDAARIAEDLAAAAASKQQPA
(ALAS) KKAGGCPFHAAQAQAQAKPAAAPKETVATADKKGK
SPRAAGGFDYEKFYEEELDKKHQDKSYRYFNNINRLA
ARFPTAHTAKVTDEVEVWC SNDYLGMGGNPVVLET
Schizophyllum MHRVLDKYGHGAGGTRNIAGNGALHLSLEQELARLH
commune H4-8 RKEGALVFTSCYVANDATLSTLGSKMPGCVIF SDRMN
HA SMIQ GIRH S GTKKVIFEHNDLADLEKKLAEYPKETP
KIIAFE S VY SMC GS IGPIKEICDLAEKYGAITFLDEVHA
Accession:
VGLYGPRGAGVAEHLDYDLHKAAGDSPDAIPGTVMD
XP 003036856.1 RVDIIT GTLGK S YGAIGGYIAGS ARF VDMIRS YAP GF IF
TT SLPPATVAGAQ A S VVYQKEYLGDRQLKQVNVREV
KRRF AELDIPVVP GP SHIVPVLVGDAALAKQASDKLL
AEHDIYVQAINYPTVARGEERLRITVTQRHTLEQMDH
LIGAVDQVFNELNINRVQDWKRLGGRASVGVPGGQD
FVEPIW TDEQVGLAD GS APL TLRNGQPNEV SHDAVV

AARSRFDWLLGPIP SHIQAKRLGQ SLEGTPIAPLAPKQ
SSGLKLPVEEMTMGQTIAVAA
-aminol evulini c MDKIARFKQTCPFLGRTKNSTLRNL STSS SPRFP SLTAL 95 acid synthase TERATKCPVMGPALNVRSKEIVAGYASVAANSDVALI
(ALAS) HKEKGVFPPPGATVEMCPHASAARAAARMADDLAA
AAEKKKGHFT SAAPRDEAAQAAAAGCPFHVKAAAD
AAAARKAAAAPAPVKAKED GGFNYE SF YVNELDKK
Crassisporium HQDKSYRYFNNINRLAAKFPVAHT SNVKDEVEVWC A
funariophilum NDYLGMGNNPVVLETMHRTLDKYGHGAGGTRNIAG
NGAMHL SLEQELATLHRKPAALVF S SCYVANDATLST
LGAKLPGCIFF SDTMNHASMIQGMRHSGAKRVLFKH
Accession:
NDLEDLENKLKQYPKDTPKVIAFESVYSMCGSIGPIKE
KAF8165006. 1 ICDLAEQYGALTFLDEVHAVGLYGPRGAGVAEHLDY
DAHVAAGESPHPIKGSVMDRVDIITGTLGKAYGAVGG
YIAGSDDF VDMIR S YAPGF IF T T SLPPATVAGARASVV
YQKHYVGDRQLKQVNVREVKRRF AELDVPVVP GP SH
IVPVLVGDAALAKAASDKLLAEHNIYVQ SINYPTVAR
GEERLRITVTPRHTLEQMDKLVRAVDKIFAELKINRLA
DWKALGGRAGVGLTAGAEEAHVDPMWTEEQLGLLD
GT SPRTLRNGEAAVVDAMAVGQ ARAVFDNLL GPI S G
KLQ SERSVLAS STPAAANPARPAARKVVKMKTGGVP
MSEDIPLPPPDVSASA
5-aminolevulinic MDKL SSL SRFKASCPFLGRTKT STLRTLCTS S SPRFP SIS 96 acid synthase IL TERATKCPVMGPALNVR SKEITAGYA S VAGS SEVD
(ALAS) QIHKQQGVTVPVNATVEMCPHASAARAAARMADDL
AAAAAQKKVGS GA S S AK AAAAGCPFHK S VAAGA S A
STASKPSAPIHKASVPGGFDYDNFYNNELEKKHKDK S
Dendrothele YRYFNNINRLASKFPVAHTGDVKDEVQVWC SNDYLG
bispora CBS MGNNPVVLETMHRTLDKYGHGAGGTRNIAGNGALH
962.96 LGLEQELAALHRKEAALVF SSCYVANDATLSTLGSKL

PGCILFSDKMNHASMIQGMRHSGAKKVIFNHNDLEDL
ENKLKQYPKETPKIIAFESVYSMCGSIGPIKEICDLAEK
Accession:
THVO5492.1 YGALTFLDEVHAVGLYGPHGAGVAEHLDYNAQKAA
GKSPEPIPGSVMDRVDIITGTLGKAYGAVGGYIAGSM
DFVDTIRSYAPGFIFTTSLPPATVSGAQASVAYQKEYL
GDRQLKQVNVREVKRRFAELDIPVIPGPSHILPVLVGD
AALAKAASDKLLTDHDIYVQSINYPTVAVGEERLRIT
VTPRHTLEQMDKLVRAVNQVFTELNINRISDWKVAG
GRAGVGMGVESVEPIWTDEQLGITDGTTPKTLRDGQR
FLVDAQGVTAARGRFDTLLGPMSGSLQANPTLPLVD
DELKVPLPTLVAAAA
5-aminolevulinic MDYAQFFNTALDRLHTERRYRVFADLERIAGRFPHAL 97 acid synthase WHSPKGKRDVVIWCSNDYLGMGQHPKVVGAMVETA
(ALAS) TRVGTGAGGTRNIAGTHHPLVQLEAELADLHGKEASL
LFTSGYVSNQTGIATIAKLIPNCLILSDELNHNSMIEGIR
Bradyrhizobium QSGCERVVFRHNDLADLEEKLKAAGPNRPKLIACESL
japonicum YSMDGDVAPLAKICDLAEKYGAMTYVDEVHAVGMY
Accession: GPRGGGIAERDGVMHRIDILEGTLAKAFGCLGGYIAA

NWERERHQDRAARVKAILNAAGLPVMSSDTHIVPLFI
GDAEKCKQASDLLLEQHGIYIQPINYPTVAKGTERLRI
TPSPYHDDGLIDQLAEALLQVWDRLGLPLKQKSLAAE
Cytochrome b5 MDKQRVFTLSQVAEHKSKQDCWIIINGRVVDVTKFLE 98 EHPGGEEVLIESAGKDATKEFQDIGHSKAAKNLLFKY
Petunia x hybrida, QIGYLQGYKASDDSELELNLVTDSIKEPNKAKEMKAY
Accession:
VIKEDPKPKYLTFVEYLLPFLAAAFYLYYRYLTGALQ
AAD10774.1 Table 12: Glossary of abbreviations Abbreviation Full Name 3GT anthocyanidin-3-0-glycotransferase 4CL 4-coumarate-CoA ligase ACC acetyl-CoA carboxylase ACOT acyl-CoA thioesterase acpP acyl carrier protein ACS acetyl-CoA synthase adhE aldehyde-alcohol dehydrogenase ADP adenosine diphosphate ALA 5-aminolevulinic acid ALAS ALA synthase ANS anthocyanin dioxygenase aroG DAHP synthase aroK shikimate kinase aroL shikimate kinase ATP adenosine triphosphate C3G cyanidin-3-0-glycoside C4H cinnimate-4-hydroxylase CHI chalcone isomerase CHS chalcone synthase CoA coenzyme A
CPR cytochrome P450 Reductase DAD diode array detector DAHP deoxy-d-arabino-heptulosonate-7-phosphate DctPQM a malonate transporter DFR dihydroflavonol 4-reductase DHK dihydrokaempferol DHM dihydromyricein DHQ dihydroquercetin DMSO dimethyl sulfoxide E4P erythrose-4-phosphate F3'H flavonoid 3' hydroxylase F3H flavanone 3-hydroxylase fabB beta-ketoacyl-ACP synthase I
fabD malonyl-coA-ACP transacylase fabF beta-ketoacyl-ACP synthase II
FadA 3-ketoacyl-CoA thiolase FadB fatty acid oxidation complex subunit alpha FadE acyl-CoA dehydrogenase GltX glutamyl-tRNA synthetase hemA glutamyl-tRNA reductase hemL glutamate-l-semialdehyde aminotransferase HPLC high performance liquid chromatography ldhA lactate dehydrogenase LAR leucoanthocyanidin reductase matB malonyl-CoA synthase matC malonate transporter mdcA malonate coA-transferase mdcC acyl-carrier protein, subunit of mdc mdcD malonyl-CoA decarboxylase, subunit of mdc mdcE co-decarboxylase, subunit of mdc pABA para-aminobenzoic acid PAL phenylalanine ammonia-lyase PanK pantothenase kinase Pdh pyruvate dehydrogenase PEP phosphoenolpyruvate pHBA para-hydroxybenzoic acid PHE phenylalanine pheA chorismate mutase / prephenate dehydrogenase poxB pyruvate dehydrogenase ppsA phosphoenolpyruvate synthase TAL tyrosine ammonia-lyase TCA tricarboxylic acid cycle tesA thioesterase I
tesB thioesterase II
tktA transketolase TRP tryptophan TYR tyrosine TyrA chorismate mutase tyrR transcriptional regulator ybgC a thioesterase yciA a thioesterase ydiB QUIN/shikamate dehydrogenase ackA-pta Acetate kinase-phosphate acetyltransferase

Claims

PCT/US2022/024591

1. An engineered host cell that comprises one or more genetic modifications resulting in production of flavonoid or anthocyanin from a carbon source that can also be an energy source, through multiple chemical intermediates, by the engineered host cell.

2. The engineered host cell of claim 1, wherein the production of flavonoid or anthocyanin from a carbon source that can also be an energy source occurs through enzymatic transformation.

3. The engineered host cell of claim 1, wherein the carbon source is selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol; and (vi) any combination thereof.

4. The engineered host cell of claim 1, wherein the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol, and (vi) any combination thereof.

5. The engineered host cell of claim 1, wherein the one or more genetic modifications lead to increase in metabolic flux to flavonoid precursors or cofactors.

6. The engineered host cell of claim 1, wherein the one or more genetic modifications cause reduction of formation of byproducts.

7. The engineered host cell of claim 1, wherein the one or more genetic modifications are at least one genetic modification selected from the group consisting of: (i) one or more modifications for over-expressing one or more endogenous genes in the engineered host cells; (ii) one or more modifications for under-expressing one or more endogenous genes in the engineered host cells; (iii) one or more genetic modification is expressing one or more non-native genes in the engineered host cells; and (iv) a combination thereof

8. The engineered host cell of claim 1, wherein the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid.

9. The engineered host cell of claim 1, wherein the engineered host cell comprises at least one or more nucleic acid sequences selected from the group consisting of: (i) nucleic acid sequences encoding tyrosine ammonia lyase activity; (ii) nucleic acid sequences encoding phenylalanine ammonia lyase activity; (iii) nucleic acid sequences encoding cinnamate 4-hydroxylase activity; (iv) nucleic acid sequences encoding 4-courmarate-CoA ligase (4CL) activity; and (v) any combination thereof

10. The engineered host cell of claim 1, wherein the engineered host cell comprises at least one or more peptides selected from the group consisting of: (i) chalcone isomerase; (ii) chalcone synthase; (iii) a fusion protein comprises a chalcone synthase and a chalcone isomerase; and (iv) any combination thereof.

11. The engineered host cell of claim 1, wherein the engineered host cell is E. coli.

12. The engineered host cell of claim 1, wherein the one or more genetic modifications decrease fatty acid biosynthesis.

13. The engineered host cell of claim 1, wherein the engineered host cell comprises an exogenous nucleic acid sequence selected from the group consisting of: (i) nucleic acid sequence encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces coumaric acid from trans-cinnamic acid; (iv) nucleic acid sequence encoding 4-courmarate-CoA ligase activity, wherein 4-courmarate-CoA ligase forms p-coumaroyl-CoA from coumaric acid (v) nucleic acid sequence encoding chalcone synthase activity, wherein chalcone synthase forms naringenin chalcone from malonyl-CoA
and p-coumaroyl-CoA; (vi) nucleic acid sequence encoding chalcone isomerase activity, wherein chalcone isomerase forms naringenin from naringenin chalcone; (vii) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (viii) any combinations thereof

14. The engineered host cell of claim 1, wherein the flavonoid is catechin.

15. A method of increasing the production of flavonoids or anthocyanins, the method comprising: providing an engineered host cell that comprises one or more genetic modifications resulting in production of flavonoid or anthocyanin from a carbon source that can also be an energy source, through multiple chemical intermediates, by the engineered host cell.

16. The method of claim 15, wherein the production of flavonoid or anthocyanin from glycerol occurs through enzymatic transformation.

17. The method of claim 15, wherein the carbon source is selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol, and (vi) any combination thereof

18. The method of claim 15, wherein the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol, and (vi) any combination thereof.

19. The method of claim 15, wherein the one or more genetic modifications lead to increase in metabolic flux to flavonoid precursors or cofactors.

20. The method of claim 15, wherein the one or more genetic modifications cause reduction in the production of byproducts.

21. The method of claim 15, wherein the one or more genetic modifications are at least one genetic modification selected from the group consisting of: (i) one or more modifications for over-expressing one or more endogenous genes in the engineered host cells; (ii) one or more modifications for under-expressing one or more endogenous genes in the engineered host cells; (iii) one or more genetic modification for expressing one or more non-native genes in the engineered host cells; and (iv) a combination thereof.

22. The method of claim 15, wherein the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid.

23. The method of claim 15, wherein the engineered host cell comprises at least one or more nucleic acid sequences selected from the group consisting of: (i) nucleic acid sequences encoding tyrosine ammonia lyase activity; (ii) nucleic acid sequences encoding phenylalanine ammonia lyase activity; (iii) nucleic acid sequences encoding cinnamate 4-hydroxylase activity; (iv) nucleic acid sequences encoding 4-courmarate-CoA ligase (4CL) activity; and (v) any combination thereof

24. The method of claim 15, wherein the engineered host cell comprises at least one or more peptides selected from the group consisting of: (i) chalcone isomerase;
(ii) chalcone synthase; (iii) a fusion protein comprises a chalcone synthase and a chalcone isomerase; and (iv) any combination thereof.

25. The method of claim 15, wherein the engineered host cell is E. coli.

26. The method of claim 15, wherein the one or more genetic modifications decrease fatty acid biosynthesis.

27. The method of claim 15, wherein the engineered host cell comprises an exogenous nucleic acid sequence selected from the group consisting of: (i) nucleic acid sequence encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acid sequence encoding 4-courmarate-CoA
ligase activity, wherein 4-courmarate-CoA ligase forms p-coumaroyl-CoA from coumaric acid (v) nucleic acid sequence encoding chalcone synthase activity, wherein chalcone synthase forms naringenin chalcone from malonyl-CoA and p-coumaroyl-CoA; (vi) nucleic acid sequence encoding chalcone isomerase activity, wherein chalcone isomerase forms naringenin from naringenin chalcone; (vii) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (viii) any combinations thereof

28. The method of claim 15, wherein the flavonoid is catechin.

29. A plurality of engineered host cells, wherein each of the plurality of the engineered host cells comprises one or more genetic modifications resulting in production of flavonoid or anthocyanin from a carbon source that can also be an energy source, through multiple chemical intermediates, by the engineered host cells.

30. The plurality of engineered host cells of claim 29, wherein the production of flavonoid or anthocyanin from glycerol occurs through enzymatic transformation.

31. The plurality of engineered host cells of claim 29, wherein the carbon source is selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol, and (vi) any combination thereof

32. The plurality of engineered host cells of claim 29, wherein the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of:
(i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol, and (vi) any combination thereof.

33. The plurality of engineered host cells of claim 29, wherein the one or more genetic modifications lead to increase in metabolic flux to flavonoid precursors or cofactors.

34. The plurality of engineered host cells of claim 29, wherein the one or more genetic modifications cause a reduction in the formation of byproducts.

35. The plurality of engineered host cells of claim 29, wherein the one or more genetic modifications are at least one genetic modification selected from the group consisting of: (i) one or more modifications for over-expressing one or more endogenous genes in the engineered host cells; (ii) one or more modifications for under-expressing one or more endogenous genes in the engineered host cells; (iii) one or more genetic modification is expressing one or more non-native genes in the engineered host cells;
and (iv) a combination thereof.

36. The plurality of engineered host cells of claim 29, wherein at least one of the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid.

37. The plurality of engineered host cells of claim 29, wherein at least one of the engineered host cell comprises at least one or more nucleic acid sequences selected from the group consisting of: (i) nucleic acid sequences encoding tyrosine ammonia lyase activity; (ii) nucleic acid sequences encoding phenylalanine ammonia lyase activity; (iii) nucleic acid sequences encoding cinnamate 4-hydroxylase activity; (iv) nucleic acid sequences encoding 4-courmarate-CoA ligase (4CL) activity; and (v) any combination thereof.

38. The plurality of engineered host cells of claim 29, wherein at least one of the engineered host cell comprises at least one or more peptides selected from the group consisting of: (i) chalcone isomerase; (ii) chalcone synthase; (iii) a fusion protein comprises a chalcone synthase and a chalcone isomerase; and (iv) any combination thereof

39. The plurality of engineered host cells of claim 29, wherein at least one of the engineered host cell is E. coli.

40. The plurality of engineered host cells of claim 29, wherein the one or more genetic modifications decrease fatty acid biosynthesis.

41. The plurality of engineered host cells of claim 29, wherein at least one of the engineered host cell comprises an exogenous nucleic acid sequence selected from the group consisting of: (i) nucleic acid sequence encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid;
(iv) nucleic acid sequence encoding 4-courmarate-CoA ligase activity, wherein 4-courmarate-CoA ligase forms p-coumaroyl-CoA from coumaric acid (v) nucleic acid sequence encoding chalcone synthase activity, wherein chalcone synthase forms naringenin chalcone from malonyl-CoA and p-coumaroyl-CoA; (vi) nucleic acid sequence encoding chalcone isomerase activity, wherein chalcone isomerase forms naringenin from naringenin chalcone; (vii) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (viii) any combinations thereof.

42. The plurality of engineered host cells of claim 29, wherein the flavonoid is catechin.

43. A method of increasing the production of flavonoids or anthocyanins, the method comprising: providing a plurality of engineered host cells, wherein each of the plurality of the engineered host cell comprises one or more genetic modifications that resulting in production of flavonoid or anthocyanin from a carbon source that can also be an energy source, through multiple chemical intermediates, through multiple chemical intermediates, by the engineered host cell.

44. The method of claim 43, wherein the one or more genetic modifications lead to increase in metabolic flux to flavonoid precursors.

45. The method of claim 43, wherein the carbon source is selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol, and (vi) any combination thereof

46. The method of claim 43, wherein the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol, and (vi) any combination thereof.

47. The method of claim 43, wherein the one or more genetic modifications lead to increase in metabolic flux to flavonoid precursors or cofactors.

48. The method of claim 43, wherein the one or more genetic modifications cause reduction in the production of byproducts.

49. The method of claim 43, wherein the one or more genetic modifications are at least one genetic modification selected from the group consisting of: (i) one or more modifications for over-expressing one or more endogenous genes in the engineered host cells; (ii) one or more modifications for under-expressing one or more endogenous genes in the engineered host cells; (iii) one or more genetic modification is expressing one or more non-native genes in the engineered host cells; and (iv) a combination thereof.

50. The method of claim 43, wherein at least one of the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid.

51. The method of claim 43, wherein at least one of the engineered host cell comprises at least one or more nucleic acid sequences selected from the group consisting of: (i) nucleic acid sequences encoding tyrosine ammonia lyase activity; (ii) nucleic acid sequences encoding phenylalanine ammonia lyase activity; (iii) nucleic acid sequences encoding cinnamate 4-hydroxylase activity; (iv) nucleic acid sequences encoding 4-courmarate-CoA ligase (4CL) activity; and (v) any combination thereof.

52. The method of claim 43, wherein at least one of the engineered host cell comprises at least one or more peptides selected from the group consisting of: (i) chalcone isomerase; (ii) chalcone synthase; (iii) a fusion protein comprises a chalcone synthase and a chalcone isomerase; and (iv) any combination thereof

53. The method of claim 43, wherein at least one of the engineered host cell is E. coli.

54. The method of claim 43, wherein the one or more genetic modifications decreases fatty acid biosynthesis.

55. The method of claim 43, wherein at least one of the engineered host cell comprises an exogenous nucleic acid sequence selected from the group consisting of: (i) nucleic acid sequence encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces coumaric acid from trans-cinnamic acid; (iv) nucleic acid sequence encoding 4-courmarate-CoA ligase activity, wherein 4-courmarate-CoA ligase forms p-coumaroyl-CoA from coumaric acid (v) nucleic acid sequence encoding chalcone synthase activity, wherein chalcone synthase forms naringenin chalcone from malonyl-CoA
and p-coumaroyl-CoA; (vi) nucleic acid sequence encoding chalcone isomerase activity, wherein chalcone isomerase forms naringenin from naringenin chalcone; (vii) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (viii) any combinations thereof

56. The method of claim 43, wherein the flavonoid is catechin.

57. An engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase the production and/or availability of malonyl-CoA.

58. The engineered host cell of claim 57, wherein the production and/or availability of malonyl-CoA is increased by transformation of acetyl-CoA to malonyl-CoA.

59. The engineered host cell of claim 57, wherein the engineered host cell comprises one or more genetic modifications selected from a group consisting of: (i) expression of acetyl-CoA carboxylase (ACC); and (ii) overexpression of acetyl-CoA
carboxylase.

60. The engineered host cell of claim 57, wherein the engineered host cell is E. coli.

61. The engineered host cell of claim 60, wherein the E. coli further comprises genes from fungi.

62. The engineered host cell of claim 59, wherein the acetyl-CoA
carboxylase is from a species selected from the group consisting of Mucor circinelloides, Rhodotorula toruloides, Lipomyces starkeyi, and Ustilago maydis, and orthologs of acetyl-CoA
carboxylase having at least 50% amino acid identity to the acetyl-CoA
carboxylase of these aforementioned species.

63. The engineered host cell of claim 57, wherein the one or more genetic modification is deletion or attenuation of one or more fatty biosynthetic genes resulting in decrease in fatty acid biosynthesis.

64. The engineered host cell of claim 57, wherein the one or more genetic modification is overexpression of acetyl-CoA synthase (AC S).

65. The engineered host cell of claim 64, wherein the acetyl-CoA synthase is selected from the group consisting of, acetyl-CoA synthase gene of E. coli, acetyl-CoA
synthase gene of Salmonella typhimurium, and orthologs of acetyl-CoA synthase gene in any other species having at least 50% amino acid identity to the acetyl-CoA synthase gene of E.
coli and Salmonella typhimurium.

66. The engineered host cell of claim 57, wherein the one or more genetic modification is selected from a group consisting of: (i) overexpression a gene encoding pyruvate dehydrogenase (PDH), wherein the PDH may include E354K mutation; (ii) exogenous nucleic acid sequence encoding a malonyl-CoA synthetase; (iii) upregulation of endogenous pantothenate kinase (PanK), wherein PanK is not feedback inhibited by coenzyme A; (iv) exogenous nucleic acid sequence encoding a malonate transporter;
and (v) any combinations thereof

67. The engineered host cell of claim 66, wherein the malonyl-CoA
synthetase is selected from the group consisting of malonyl-CoA synthetases of Streptomyces coelicolor, Rhodopseudomonas palustris, or a malonyl-CoA synthetase having at least 50%
identity to any of these or other naturally occurring malonyl-CoA synthetases.

68. The engineered host cell of claim 63, wherein the wherein one or more genetic modifications to decrease fatty acid biosynthesis is selected from the group consisting of: (i) mutation or downregulation of a gene encoding malonyl-CoA-ACP
transacylase (E. coli fabD); (ii) modifications to the gene beta-ketoacyl-ACP synthase II
(E. coli fabF); (iii) downregulation of beta-ketoacyl-ACP synthase I enzyme (E. coli fabB); (iv) downregulation of acyl carrier protein (E. coli acpP); and (v) any combinations thereof.

69. The engineered host cell of claim 57, wherein the engineered host cell comprises peptides selected from a group consisting of: (i) acetyl-CoA carboxylase (ACC) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID
NO: 15 or SEQ ID NO: 16; (ii) malonate CoA-transferase having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 19;
(iii) acetyl-CoA synthase (ACS) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 16; (iv) malonyl-CoA synthase having an amino acid sequence at least 80% identical SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID
NO:
79; (v) malonate transporter having an amino acid sequence at least 80%
identical to SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, or SEQ ID NO: 87; (vi) pantothenate kinase having an amino acid sequence at least 80% identical to SEQ ID NO: 88, SEQ ID NO: 89, or SEQ ID NO: 90; and (vii) any combinations thereof.

70. A method of increasing the production of flavonoids comprising an engineered host cell, wherein the one or more engineered host cells comprise one or more genetic modifications to increase the production and/or availability of malonyl-CoA.

71. The method of claim 70, wherein the production and/or availability of malonyl-CoA is increased by transformation of acetyl-CoA to malonyl-CoA.

72. The method of claim 70, wherein the engineered host cell comprises one or more genetic modifications selected from a group consisting of: (i) expression of acetyl-CoA
carboxylase (ACC); and (ii) overexpression of acetyl-CoA carboxylase.

73. The method of claim 70, wherein the engineered host cell is E. coli.

74. The method of claim 73, wherein the E. coli further comprises genes from fungi.

75. The method of claim 70, wherein the acetyl-CoA carboxylase is from a species selected from the group consisting of Mucor circinelloides, Rhodotorula toruloides, Lipomyces starkeyi, and Ustilago maydis, and orthologs of acetyl-CoA carboxylase having at least 50% amino acid identity to the acetyl-CoA carboxylase of these aforementioned species.

76. The method of claim 70, wherein the one or more genetic modification is deletion or attenuation of one or more fatty biosynthetic genes resulting in decrease in fatty acid biosynthesis.

77. The method of claim 70, wherein the one or more genetic modification is overexpression of acetyl-CoA synthase (ACS).

78. The method of claim 77, wherein the acetyl-CoA synthase (ACS) is selected from the group consisting of, acetyl-CoA synthase gene of E. coli, acetyl-CoA synthase gene of Salmonella typhimurium, and orthologs of acetyl-CoA synthase gene in any other species having at least 50% amino acid identity to the acetyl-CoA synthase gene of E.
coli and Salmonella typhimurium.

79. The method of claim 14, wherein the one or more genetic modification is selected from a group consisting of: (i) overexpression a gene encoding pyruvate dehydrogenase (PDH), wherein the PDH may include E354K mutation; (ii) exogenous nucleic acid sequence encoding a malonyl-CoA synthetase; (iii) upregulation of endogenous pantothenate kinase (PanK), wherein PanK is not feedback inhibited by coenzyme A;
(iv) exogenous nucleic acid sequence encoding a malonate transporter; and (v) any combinations thereof

80. The method of claim 79, wherein the malonyl-CoA synthetase is selected from the group consisting of malonyl-CoA synthetases of Streptomyces coelicolor, Rhodopseudomonas palustris, or a malonyl-CoA synthetase having at least 50%
identity to any of these or other naturally occurring malonyl-CoA synthetases.

81. The method of claim 76, wherein the wherein one or more genetic modifications to decrease fatty acid biosynthesis is selected from the group consisting of: (i) mutation or downregulation of a gene encoding malonyl-CoA-ACP transacylase (E. coli fabD);
(ii) modifications to the gene beta-ketoacyl-ACP synthase II (E. coli fabF); (iii) downregulation of beta-ketoacyl-ACP synthase I enzyme (E. coli fabB); (iv) downregulation of acyl carrier protein (E. coli acpP); and (v) any combinations thereof.

82. The method of claim 70, wherein the engineered host cell comprises peptides selected from a group consisting of: (i) acetyl-CoA carboxylase (ACC) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 15 or SEQ
ID NO: 16; (ii) malonate CoA-transferase having an amino acid sequence at least 80%
identical to the polypeptide set forth in SEQ ID NO: 19; (iii) acetyl-CoA
synthase (ACS) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 16; (iv) malonyl-CoA synthase having an amino acid sequence at least 80% identical SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 79; (v) malonate transporter having an amino acid sequence at least 80% identical to SEQ ID NO:
80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, or SEQ ID NO: 87; (vi) pantothenate kinase having an amino acid sequence at least 80% identical to SEQ ID NO: 88, SEQ ID NO: 89, or SEQ ID NO:

90; and (vii) any combinations thereof

83. An engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase endogenous biosynthesis of tyrosine.

84. The engineered host cell of claim 83, wherein the one or more genetic modifications comprises upregulation of 3-deoxy-D-arabino-heptulosonate synthase.

85. The engineered host cell of claim 83, wherein the one or more genetic modifications are selected from a group consisting of: (i) upregulation of chorismate mutase;
(ii) upregulation of prephenate dehydrogenase; (iii) overexpression of shikimate kinase;
(iv) overexpression of shikimate dehydrogenase; and (v) any combinations thereof.

86. The engineered host cell of claim 83, wherein the one or more genetic modifications comprises downregulation of L-phenylalanine biosynthetic pathway.

87. The engineered host cell of claim 83, wherein the one or more genetic modifications comprises expression of exogenous phosphoenolpyruvate synthase (ppsA).

88. The engineered host cell of claim 83, wherein the one or more genetic modifications comprises expression of exogenous transketolase (tktA).

89. The engineered host cell of claim 83, wherein the one or more genetic modifications comprises disruption of tyrR gene.

90. The engineered host of claim 83, wherein the one or more genetic modifications are selected from a group consisting of: (i) expression or overexpression of (D146N) variant of phospho-2-dehydro-3-deoxyheptonate aldolase; (ii) expression or overexpression of variant of 3-dehydroquinate synthase (aroB); (iii) overexpression of transketolase tktA; (iv) deletion of shikimate kinase (aroK); (v) deletion of tyrR; (vi) expression or overexpression of A354V variant of chorismate mutase (tyrA);
(vi) and any combination thereof

91. A method of increasing endogenous biosynthesis of tyrosine comprising an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase endogenous biosynthesis of tyrosine.

92. The method of claim 91, wherein the one or more genetic modifications comprises upregulation of 3-deoxy-D-arabino-heptulosonate synthase.

93. The method of claim 91, wherein the one or more genetic modifications are selected from a group consisting of: (i) upregulation of chorismate mutase; (ii) upregulation of prephenate dehydrogenase; (iii) overexpression of shikimate kinase; (iv) overexpression of shikimate dehydrogenase; and (v) any combinations thereof

94. The method of claim 91, wherein the one or more genetic modifications comprises downregulation of L-phenylalanine biosynthetic pathway.

95. The method of claim 91, wherein the one or more genetic modifications comprises expression of exogenous phosphoenolpyruvate synthase (ppsA).

96. The method of claim 91, wherein the one or more genetic modifications comprises expression of exogenous transketolase (tktA).

97. The method of claim 91, wherein the one or more genetic modifications comprises disruption of tyrR gene.

98. The method of claim 91, wherein the one or more genetic modifications are selected from a group consisting of: (i) expression or overexpression of (D146N) variant of phospho-2-dehydro-3-deoxyheptonate aldolase; (ii) expression or overexpression of variant of 3-dehydroquinate synthase (aroB); (iii) overexpression of transketolase tktA;

(iv) deletion of shikimate kinase (aroK); (v) deletion of tyrR; (vi) expression or overexpression of A354V variant of chorismate mutase (tyrA); (vi) and any combination thereof.

99. An engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase transformation of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G).

100. The engineered host cell of claim 99, wherein the one or more genetic modifications comprising overexpression of anthocyanin synthase.

101. The engineered host cell of claim 100, wherein the anthocyanin synthase is selected from a group consisting of: (i) anthocyanin synthase of Carica papaya (SEQ. ID

NO:13); (ii) the anthocyanin synthase has an amino acid sequence at least 80%
identical to SEQ. ID NO: 66, SEQ. ID NO: 67, SEQ. ID NO: 68, or SEQ. ID NO:
69;
(iii) the anthocyanin synthase has an amino acid sequence at least 80%
identical to SEQ. ID NO: 13; and (iv) any combinations thereof.

102. The engineered host cell of claim 100, wherein the engineered host cell further comprises flavonoid-3-glucosyl transferase (3GT).

103. The engineered host cell of claim 102, wherein the flavonoid-3-glucosyl transferase is selected from a group consisting of: (i) flavonoid-3-glucosyl transferase in Vitis labrusca (SEQ. ID NO:14); (ii) the flavonoid-3-glucosyl transferase has an amino acid sequence at least 80% identical to SEQ. ID NO: 70, SEQ. ID NO: 71, SEQ. ID NO:
72, or SEQ. ID NO: 73; and (iii) any combinations thereof.

104. A method of increasing the production of flavonoids comprising an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase transformation of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G).

105. The method of claim 104, wherein the one or more genetic modifications comprising overexpression of anthocyanin synthase.

106. The method of claim 104, wherein the anthocyanin synthase is selected from a group consisting of: (i) anthocyanin synthase of Carica papaya (SEQ. ID NO:13); (ii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ.
ID
NO: 66, SEQ. ID NO: 67, SEQ. ID NO: 68, or SEQ. ID NO: 69; (iii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 13;
and (iv) any combinations thereof.

107. The method of claim 105, wherein the engineered host cell further comprises flavonoid-3-glucosyl transferase (3GT).

108. The method of claim 106, wherein the flavonoid-3-glucosyl transferase is selected from a group consisting of: (i) flavonoid-3-glucosyl transferase in Vitis labrusca (SEQ. ID
NO:14); (ii) the flavonoid-3-glucosyl transferase has an amino acid sequence at least 80% identical to SEQ. ID NO: 70, SEQ. ID NO: 71, SEQ. ID NO: 72, or SEQ. ID
NO:
73; and (iii) any combinations thereof

109. A method of increasing the transformation of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G) comprising anthocyanin synthase, wherein the anthocyanin synthase is selected from a group consisting of: (i) anthocyanin synthase of Carica papaya (SEQ. ID NO:13); (ii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 66, SEQ. ID NO: 67, SEQ. ID NO: 68, or SEQ. ID
NO:
69; (iii) the anthocyanin synthase has an amino acid sequence at least 80%
identical to SEQ. ID NO: 13; and (iv) any combinations thereof.

110. A method of increasing the transformation of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G) comprising flavonoid-3-glucosyl transferase (3GT), wherein the flavonoid-3-glucosyl transferase is selected from a group consisting of: (i) flavonoid-3-glucosyl transferase in Vitis labrusca (SEQ. ID NO:14); (ii) the flavonoid-3-glucosyl transferase has an amino acid sequence at least 80% identical to SEQ. ID NO:
70, SEQ.
ID NO: 71, SEQ. ID NO: 72, or SEQ. ID NO: 73; and (iii) any combinations thereof.

111. An engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase the production of dihydroquercetin (DHQ), dihydromyricein (DHIVI), eriodictoyl (EDL), and/or pentahydroxyflayaone (PHF), wherein the engineered host cell comprises cytochrome P450 reductase (CPR) and at least one of flavanone-3-hydroxylase (F3H), flavanone-3'-hydroxylase (F3'H), or flavonoid 3',5'-hydroxylase (F3'5'H).

112. The cell of claim 111, wherein the precursor for increase in production of dihydroquercetin (DHQ), dihydromyricein (DHIVI), eriodictoyl (EDL), and/or pentahydroxyflayanone (PHF) is naringenin and/or dihydrokaempferol (DHK).

113. The engineered host cell of claim 111, wherein the flavonoid 3'-hydroxylase (F3'H) is truncated to remove the N-terminal leader sequence.

114. The engineered host cell of claim 111, wherein the flavonoid 3',5'-hydroxylase (F3'5'H) is truncated to remove the N-terminal leader sequence.

115. The engineered host cell of claim 111, wherein the cytochrome P450 reductase (CPR) is truncated to remove the N-terminal leader sequence.

116. The engineered host cell of claim 111, wherein the flavonoid 3'-hydroxylase (F3'H) is fused with cytochrome P450 reductase (CPR).

117. The engineered host cell of claim 111, wherein the flavonoid 3',5'-hydroxylase (F3'5'H) is fused with cytochrome P450 reductase (CPR).

118. The engineered host cell of claim 111, wherein the flavanone-3'-hydroxylase (F3'H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ
ID NO. 8.

119. The engineered host cell of claim 111, wherein the cytochrome P450 reductase (CPR) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ
ID NO. 9.

120. The engineered host cell of claim 111, wherein the flavonoid 3',5'-hydroxylase (F3'5'H) has an amino acid sequence at least 80% identical to the polypeptides selected from a group consisting of: (i) SEQ ID NO. 10, (ii) SEQ ID NO. 56, and (iii) SEQ ID
NO. 57.

121. The engineered host cell of claim 111, wherein the engineered host cell further comprises cytochrome bs.

122. The engineered host cell of claim 121, wherein the cytochrome bs has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 98.

123. The engineered host cell of claim 111, wherein the flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to the polypeptides selected from a group consisting of: (i) SEQ ID NO. 7, (ii) SEQ ID NO. 45, (iii) SEQ ID NO. 46, (iv) SEQ ID
NO. 47, and (v) SEQ ID NO. 48.

124. A method of increasing the production of dihydroquercetin (DHQ), dihydromyricein (DHIVI), eriodictoyl (EDL), and/or pentahydroxyflayaone (PHF) comprising an engineered host cell, wherein the engineered host cell comprises cytochrome reductase (CPR) and at least one of flavanone-3-hydroxylase (F3H), flavanone-3'-hydroxylase (F3'H), or flavonoid 3',5'-hydroxylase (F3'5'H).

125. The method of claim 124, wherein the precursor for increase in production of dihydroquercetin (DHQ), dihydromyricein (DHIVI), eriodictoyl (EDL), and/or pentahydroxyflayanone (PHF) are naringenin and/or dihydrokaempferol (DHK).

126. The method of claim 124, wherein the flavonoid 3'-hydroxylase (F3'H) is truncated to remove the N-terminal leader sequence.